1 00:00:00,500 --> 00:00:02,940 The following content is provided under a Creative 2 00:00:02,940 --> 00:00:04,300 Commons license. 3 00:00:04,300 --> 00:00:06,780 Your support will help MIT OpenCourseWare 4 00:00:06,780 --> 00:00:11,140 continue to offer high-quality educational resources for free. 5 00:00:11,140 --> 00:00:13,770 To make a donation or view additional materials 6 00:00:13,770 --> 00:00:17,720 from hundreds of MIT courses, visit MIT Open CourseWare 7 00:00:17,720 --> 00:00:18,690 at ocw.mit.edu. 8 00:00:23,714 --> 00:00:25,130 JOANNE STUBBE: We talked last time 9 00:00:25,130 --> 00:00:28,580 about kinetics, steady-state kinetics, 10 00:00:28,580 --> 00:00:32,330 pre-steady-state kinetics, how you design the experiments, 11 00:00:32,330 --> 00:00:35,490 what kinds of information you can get out 12 00:00:35,490 --> 00:00:37,790 of each experimental design. 13 00:00:37,790 --> 00:00:40,790 And we introduced all of that material. 14 00:00:40,790 --> 00:00:43,945 And today, what I want to do is come back to the model. 15 00:00:43,945 --> 00:00:45,320 You saw it at the very beginning, 16 00:00:45,320 --> 00:00:47,460 and you've seen it in a lecture. 17 00:00:47,460 --> 00:00:51,850 And specifically, where did this model come from? 18 00:00:51,850 --> 00:00:53,350 That's what we're going to focus on. 19 00:00:53,350 --> 00:00:57,290 OK, and so in order to be able to understand this model, 20 00:00:57,290 --> 00:00:58,409 you have to design assays. 21 00:00:58,409 --> 00:01:00,200 And you're going to see over and over again 22 00:01:00,200 --> 00:01:02,033 over the course of the semester figuring out 23 00:01:02,033 --> 00:01:04,940 how to design an assay, in this case, isn't so hard, 24 00:01:04,940 --> 00:01:07,250 but in many cases is really tough. 25 00:01:07,250 --> 00:01:10,760 And that's the key to being able to get kinetic information 26 00:01:10,760 --> 00:01:12,240 is designing the assay. 27 00:01:12,240 --> 00:01:14,180 So if you look here, today, we're 28 00:01:14,180 --> 00:01:18,560 going to be looking at GTP is hydrolyzed. 29 00:01:18,560 --> 00:01:21,770 So you need to think about, as a chemist, how 30 00:01:21,770 --> 00:01:23,914 you could study that reaction. 31 00:01:23,914 --> 00:01:25,580 How would you look at starting material? 32 00:01:25,580 --> 00:01:27,800 How would you look at product as a function 33 00:01:27,800 --> 00:01:30,870 of time, which is what we were talking about last time? 34 00:01:30,870 --> 00:01:33,290 And we're going to talk about that first. 35 00:01:33,290 --> 00:01:37,325 We're going to talk about use of radioisotopes first. 36 00:01:37,325 --> 00:01:41,690 And we've already been talking about radioisotopes in class 37 00:01:41,690 --> 00:01:43,170 the last couple of lectures. 38 00:01:43,170 --> 00:01:46,310 So we decided to focus most of our energy 39 00:01:46,310 --> 00:01:47,900 now on radioisotopes. 40 00:01:47,900 --> 00:01:50,840 And then the second kind of probe you're going to see 41 00:01:50,840 --> 00:01:52,340 is a fluorescent probe. 42 00:01:52,340 --> 00:01:54,970 We're going to use fluorescent probes over and over again. 43 00:01:54,970 --> 00:01:57,669 And the details of the fluorescent probes 44 00:01:57,669 --> 00:01:59,210 and how they work isn't going to come 45 00:01:59,210 --> 00:02:02,820 in until the last recitation, which is recitation 13. 46 00:02:02,820 --> 00:02:05,780 So from the point of view of thinking 47 00:02:05,780 --> 00:02:08,330 about Rodnina's paper, what you need 48 00:02:08,330 --> 00:02:10,639 to think about is, if you have a probe, 49 00:02:10,639 --> 00:02:14,370 and you stick it in a different environment, it changes. 50 00:02:14,370 --> 00:02:17,701 And you can watch it change, OK, without looking at the details. 51 00:02:17,701 --> 00:02:19,700 But that's something you do need to think about, 52 00:02:19,700 --> 00:02:21,550 but we're not going to talk about that. 53 00:02:21,550 --> 00:02:27,350 OK, so we have a way of monitoring potentially GTPase. 54 00:02:27,350 --> 00:02:29,910 And we'll talk about that today. 55 00:02:29,910 --> 00:02:32,600 What other reaction can we monitor in here? 56 00:02:32,600 --> 00:02:37,790 We can monitor formation of the polypeptide chain. 57 00:02:37,790 --> 00:02:39,170 And so that's the other thing. 58 00:02:39,170 --> 00:02:42,890 And both of these chemical transformations 59 00:02:42,890 --> 00:02:44,335 use radioactivity. 60 00:02:44,335 --> 00:02:47,930 OK, so that's where we're going to focus on it initially. 61 00:02:47,930 --> 00:02:50,480 And then hopefully-- how many of you 62 00:02:50,480 --> 00:02:54,890 went back and reread the paper for this week from last week? 63 00:02:54,890 --> 00:02:58,200 Did any of you go back and reread it? 64 00:02:58,200 --> 00:02:59,900 OK, so I think it's good. 65 00:02:59,900 --> 00:03:02,690 I just think, you know, every time I read a paper-- 66 00:03:02,690 --> 00:03:03,350 I read a paper. 67 00:03:03,350 --> 00:03:07,190 Sometimes, I've read it 10, 15 times over the course 68 00:03:07,190 --> 00:03:08,010 of my career. 69 00:03:08,010 --> 00:03:11,450 And as I learn more and think about things differently, 70 00:03:11,450 --> 00:03:13,490 I keep seeing new things. 71 00:03:13,490 --> 00:03:16,790 And this paper is just packed full of information. 72 00:03:16,790 --> 00:03:19,610 So I could say you could read it another 10 times, 73 00:03:19,610 --> 00:03:22,430 and you'd still keep learning stuff out of reading it. 74 00:03:22,430 --> 00:03:24,099 And in the very beginning, that's 75 00:03:24,099 --> 00:03:25,640 what we're trying to teach you to do. 76 00:03:25,640 --> 00:03:28,280 What do you look at in the paper to learn 77 00:03:28,280 --> 00:03:30,440 how to critically evaluate what's 78 00:03:30,440 --> 00:03:32,180 being presented in the model, which 79 00:03:32,180 --> 00:03:36,050 is maybe what you're going to build your research program on? 80 00:03:36,050 --> 00:03:38,750 Somebody else's data, is it correct? 81 00:03:38,750 --> 00:03:39,625 Is it not correct? 82 00:03:39,625 --> 00:03:42,299 OK, so we're going to use radioactivity. 83 00:03:42,299 --> 00:03:43,340 I'm going to start there. 84 00:03:43,340 --> 00:03:47,450 And then to look at these first few steps, 85 00:03:47,450 --> 00:03:49,640 which are binding steps, that's where 86 00:03:49,640 --> 00:03:52,070 we're going to look at the fluorescent probe. 87 00:03:52,070 --> 00:03:53,570 And there were three different kinds 88 00:03:53,570 --> 00:03:57,140 of experiments that were described in this paper-- 89 00:03:57,140 --> 00:04:00,740 looking at the rates of the reactions as a function 90 00:04:00,740 --> 00:04:02,374 of the concentration of the ribosome-- 91 00:04:02,374 --> 00:04:04,790 you need to think about why they looked at a concentration 92 00:04:04,790 --> 00:04:06,200 dependence-- 93 00:04:06,200 --> 00:04:09,350 measuring fluorescence changes, and then they 94 00:04:09,350 --> 00:04:13,200 used non-hydrolyzable GTP analog. 95 00:04:13,200 --> 00:04:16,066 Why did they use that? 96 00:04:16,066 --> 00:04:18,810 Do you remember what the non-hydrolyzable GDP 97 00:04:18,810 --> 00:04:22,060 analog was? 98 00:04:22,060 --> 00:04:24,480 So where's the n? 99 00:04:24,480 --> 00:04:26,604 AUDIENCE: It's between beta and gamma. 100 00:04:26,604 --> 00:04:28,270 JOANNE STUBBE: So it's non-hydrolyzable. 101 00:04:28,270 --> 00:04:30,400 It is hydrolyzable, but not under 102 00:04:30,400 --> 00:04:31,670 the experimental conditions. 103 00:04:31,670 --> 00:04:33,050 So what does it do? 104 00:04:33,050 --> 00:04:34,660 Why would you want to use something 105 00:04:34,660 --> 00:04:37,225 like that to get information about the first few steps? 106 00:04:39,840 --> 00:04:41,674 AUDIENCE: It's along the reaction continuum. 107 00:04:41,674 --> 00:04:43,048 JOANNE STUBBE: Yeah, so you don't 108 00:04:43,048 --> 00:04:44,260 let the reaction continue. 109 00:04:44,260 --> 00:04:47,110 So what that does, if it's working correctly, 110 00:04:47,110 --> 00:04:48,960 is it puts a block here. 111 00:04:48,960 --> 00:04:54,050 And then you can potentially monitor what's going on here. 112 00:04:54,050 --> 00:04:56,820 And from the data that you looked at, 113 00:04:56,820 --> 00:04:59,520 it's not really so clear what was going on there 114 00:04:59,520 --> 00:05:02,480 unless you went back and read the preceding paper. 115 00:05:02,480 --> 00:05:05,370 So there had been a decade worth of experiments on this system 116 00:05:05,370 --> 00:05:08,500 before this paper came out summarizing 117 00:05:08,500 --> 00:05:13,380 the conclusions about what they are thinking about fidelity. 118 00:05:13,380 --> 00:05:16,830 OK, so what we're going to do is talk about radioactivity. 119 00:05:16,830 --> 00:05:19,590 And our objective is simply-- and we'll come back to this 120 00:05:19,590 --> 00:05:21,190 at the very end-- 121 00:05:21,190 --> 00:05:26,370 is to use all this experimental data, the concentration 122 00:05:26,370 --> 00:05:29,400 dependence, the radioactive isotope experiments, 123 00:05:29,400 --> 00:05:32,160 the stop flow fluorescence experiments, 124 00:05:32,160 --> 00:05:34,680 and try to come up with a model that 125 00:05:34,680 --> 00:05:36,860 can explain all of the data. 126 00:05:36,860 --> 00:05:39,690 OK, so you make some measurement. 127 00:05:39,690 --> 00:05:42,700 What you're measuring is some k apparent. 128 00:05:42,700 --> 00:05:45,180 And that's usually a first-order rate constant 129 00:05:45,180 --> 00:05:47,310 because it's happening on the enzyme. 130 00:05:47,310 --> 00:05:48,950 OK, so you measure these numbers. 131 00:05:48,950 --> 00:05:49,950 Well, what do they mean? 132 00:05:49,950 --> 00:05:51,290 You don't know what they mean. 133 00:05:51,290 --> 00:05:52,873 And why don't you know what they mean? 134 00:05:52,873 --> 00:05:55,270 Because the kinetic mechanism is so complicated. 135 00:05:55,270 --> 00:05:57,990 You saw that with the steady-state analysis 136 00:05:57,990 --> 00:06:00,520 of km and kcat last time. 137 00:06:00,520 --> 00:06:02,700 So in the end, though, if you come up with a model, 138 00:06:02,700 --> 00:06:05,130 and it can explain all the data because you've 139 00:06:05,130 --> 00:06:07,440 done many, many experiments, it can 140 00:06:07,440 --> 00:06:11,370 be quite informative about the question we're 141 00:06:11,370 --> 00:06:16,620 focused on is specificity. 142 00:06:16,620 --> 00:06:21,090 How do you distinguish between phenylalanine and leucine 143 00:06:21,090 --> 00:06:22,710 and proofreading? 144 00:06:22,710 --> 00:06:24,372 How do you decide whether you're going 145 00:06:24,372 --> 00:06:30,570 to form the right peptide bond or the incorrectly charged tRNA 146 00:06:30,570 --> 00:06:31,950 is going to dissociate? 147 00:06:31,950 --> 00:06:34,110 OK, so that's what you want to come out with. 148 00:06:34,110 --> 00:06:37,950 You want to look at the ratio of these rate constants 149 00:06:37,950 --> 00:06:41,350 and the ratio k3 to k minus 2. 150 00:06:41,350 --> 00:06:44,570 And when you look at the experimental data, which we'll 151 00:06:44,570 --> 00:06:48,300 look at the end today, it should make sense to you 152 00:06:48,300 --> 00:06:49,780 in terms of this model. 153 00:06:49,780 --> 00:06:51,870 OK, but let's put it this way. 154 00:06:51,870 --> 00:06:54,360 In most cases, you don't come out with a unique model. 155 00:06:54,360 --> 00:06:57,990 It's a working hypothesis that people for the next 15 years, 156 00:06:57,990 --> 00:07:00,960 if it's an interesting problem, will take pot shots at 157 00:07:00,960 --> 00:07:05,850 to try to understand in more detail what's really going on. 158 00:07:05,850 --> 00:07:11,640 OK, so what I want to do is talk about two methods, 159 00:07:11,640 --> 00:07:15,330 but the focus probably won't get very far 160 00:07:15,330 --> 00:07:16,560 in terms of the second one. 161 00:07:16,560 --> 00:07:20,460 But today, we're going to look at radioisotopes and how 162 00:07:20,460 --> 00:07:24,480 you use that to do the assay for GDP hydrolysis 163 00:07:24,480 --> 00:07:27,230 and peptide bond formation. 164 00:07:27,230 --> 00:07:30,940 OK, so what is an isotope? 165 00:07:30,940 --> 00:07:34,800 OK, so how many of you guys have actually 166 00:07:34,800 --> 00:07:36,420 worked with radioisotopes? 167 00:07:36,420 --> 00:07:38,580 Any of you? 168 00:07:38,580 --> 00:07:41,530 No, OK, so you know, maybe they don't use this anymore. 169 00:07:41,530 --> 00:07:44,190 Biochemists for the decades have used isotopes. 170 00:07:44,190 --> 00:07:46,620 Every paper I read has isotopes in it. 171 00:07:46,620 --> 00:07:48,760 But you know, I'm old school. 172 00:07:48,760 --> 00:07:50,400 So maybe people don't use it. 173 00:07:50,400 --> 00:07:53,880 But I think the power of it is its sensitivity. 174 00:07:53,880 --> 00:07:56,090 I'm going to show you that today. 175 00:07:56,090 --> 00:07:58,330 And the other power of it is that you 176 00:07:58,330 --> 00:08:00,570 have no perturbation of your system 177 00:08:00,570 --> 00:08:02,640 and there are almost no probes like that. 178 00:08:02,640 --> 00:08:04,810 You're sticking on green fluorescent protein. 179 00:08:04,810 --> 00:08:07,710 Well, what does it do to the whole rest of the protein? 180 00:08:07,710 --> 00:08:11,310 You have to perturb to see, but radioisotopes 181 00:08:11,310 --> 00:08:12,570 have minimal perturbation. 182 00:08:12,570 --> 00:08:16,710 So it's still a very important probe, 183 00:08:16,710 --> 00:08:19,140 but it probably depends on what kinds of questions 184 00:08:19,140 --> 00:08:20,200 you're focused on. 185 00:08:20,200 --> 00:08:24,286 So what is an isotope? 186 00:08:24,286 --> 00:08:36,720 So an isotope is atoms with the same number of protons 187 00:08:36,720 --> 00:08:38,349 and a different number of neutrons. 188 00:08:44,260 --> 00:08:48,160 That's called the mass number. 189 00:08:48,160 --> 00:08:50,500 So what you have here for carbon, 190 00:08:50,500 --> 00:08:53,490 which is one of the common isotopes you guys will be using 191 00:08:53,490 --> 00:08:55,580 if you do any kind of biochemistry, 192 00:08:55,580 --> 00:08:58,790 we have C-12, C-13, and C-14. 193 00:08:58,790 --> 00:09:03,240 OK, and so this is the atomic number, which 194 00:09:03,240 --> 00:09:04,830 is the number of protons. 195 00:09:04,830 --> 00:09:10,440 OK, so the only difference between these guys 196 00:09:10,440 --> 00:09:12,890 is a neutron or two neutrons. 197 00:09:12,890 --> 00:09:16,500 OK, so there's minimal difference. 198 00:09:16,500 --> 00:09:20,790 And so what are the isotopes that you see used in biology? 199 00:09:20,790 --> 00:09:23,460 So we've already seen many of these in this paper, 200 00:09:23,460 --> 00:09:27,750 but we've also talked about some of them in class today 201 00:09:27,750 --> 00:09:29,110 and in the preceding class. 202 00:09:29,110 --> 00:09:33,630 So we're going to be using over the course of the semester 203 00:09:33,630 --> 00:09:35,760 isotopes of hydrogen. Why? 204 00:09:35,760 --> 00:09:38,340 Because if you look at your metabolic pathways, 205 00:09:38,340 --> 00:09:40,505 you're always cleaving carbon-hydrogen bonds. 206 00:09:40,505 --> 00:09:45,030 OK, so this isotope becomes incredibly important. 207 00:09:45,030 --> 00:09:51,286 C-12, C-13, anybody know where you use C-13? 208 00:09:51,286 --> 00:09:52,194 AUDIENCE: In NMR. 209 00:09:52,194 --> 00:09:55,050 JOANNE STUBBE: NMR, so if you're working for Mei Hong, 210 00:09:55,050 --> 00:09:59,490 you might be doing isotopic labeling using C-13. 211 00:09:59,490 --> 00:10:03,000 If you're doing any kind of metabolic label chasing, 212 00:10:03,000 --> 00:10:05,490 you're going to see the radioisotope is, which is what 213 00:10:05,490 --> 00:10:06,780 we're talking about, is C-14. 214 00:10:06,780 --> 00:10:08,540 Working 215 00:10:08,540 --> 00:10:13,320 So you see often, all the time, you see nitrogen and oxygen. 216 00:10:13,320 --> 00:10:15,960 And oxygen has three isotopes. 217 00:10:15,960 --> 00:10:17,400 Nitrogen has two. 218 00:10:17,400 --> 00:10:19,043 None of them are radioactive. 219 00:10:19,043 --> 00:10:22,500 OK, so you're never going to be using the methods 220 00:10:22,500 --> 00:10:24,450 we're describing today. 221 00:10:24,450 --> 00:10:27,180 But frequently, in NMR again, you 222 00:10:27,180 --> 00:10:31,250 might replace N-14 with an N-15. 223 00:10:31,250 --> 00:10:33,600 And today, we will see that we're 224 00:10:33,600 --> 00:10:36,840 using isotopes of phosphorus. 225 00:10:36,840 --> 00:10:38,510 What about phosphorus-31? 226 00:10:38,510 --> 00:10:41,404 Where do you see that? 227 00:10:41,404 --> 00:10:42,570 Have you thought about this? 228 00:10:42,570 --> 00:10:44,220 Maybe you have, and maybe you haven't. 229 00:10:44,220 --> 00:10:49,090 Phosphorus-31 versus phosphorus-32, 230 00:10:49,090 --> 00:10:52,790 what's the normal abundance isotope of phosphorus? 231 00:10:52,790 --> 00:10:56,330 31, so phosphorus-31 has a nuclear spin of a 1/2. 232 00:10:56,330 --> 00:10:59,660 So you frequently use that as well in NMR. 233 00:10:59,660 --> 00:11:02,105 And P-32 is used-- 234 00:11:02,105 --> 00:11:05,180 it's radioactive and is used in today's experiments. 235 00:11:05,180 --> 00:11:07,970 OK, so this is something that in the back of your mind 236 00:11:07,970 --> 00:11:09,080 you should think about. 237 00:11:09,080 --> 00:11:12,440 What are stable versus unstable isotopes? 238 00:11:12,440 --> 00:11:16,530 And what we're talking about today is unstable isotopes. 239 00:11:16,530 --> 00:11:18,860 So what I want to do is we're not 240 00:11:18,860 --> 00:11:20,570 going to go into this in a lot of detail, 241 00:11:20,570 --> 00:11:22,997 but I want to describe the things I think 242 00:11:22,997 --> 00:11:24,830 you need to think about if you're ever going 243 00:11:24,830 --> 00:11:28,610 to use radioactivity and how you make measurements, 244 00:11:28,610 --> 00:11:30,210 quantitative measurements. 245 00:11:30,210 --> 00:11:33,830 And so we're going to be looking at a radioisotope. 246 00:11:36,470 --> 00:11:38,867 And what do we know about radioisotopes? 247 00:11:38,867 --> 00:11:39,575 They're unstable. 248 00:11:42,776 --> 00:11:48,680 OK, and depending on which atoms they are, 249 00:11:48,680 --> 00:11:51,740 they have different stabilities. 250 00:11:51,740 --> 00:11:57,260 And they decay spontaneously into some new configuration. 251 00:11:57,260 --> 00:12:08,960 They have a nuclear decay spontaneously into a new state. 252 00:12:11,930 --> 00:12:16,230 And during this process, during this decay, 253 00:12:16,230 --> 00:12:18,950 they emit ionizing radiation. 254 00:12:18,950 --> 00:12:20,730 They emit energy. 255 00:12:20,730 --> 00:12:23,280 So during this process-- 256 00:12:23,280 --> 00:12:25,395 so this is the whole thing you need to remember. 257 00:12:29,930 --> 00:12:32,200 They emit energy. 258 00:12:32,200 --> 00:12:36,680 And the energy is in the form of ionizing radiation. 259 00:12:36,680 --> 00:12:40,420 It could be alpha, beta, or gamma radiation. 260 00:12:40,420 --> 00:12:42,400 And so what we're going to be looking for 261 00:12:42,400 --> 00:12:47,280 is trying to detect that energy that's actually released. 262 00:12:47,280 --> 00:12:50,470 OK, so before I go on, we've already 263 00:12:50,470 --> 00:12:53,590 seen all of these isotopes used already 264 00:12:53,590 --> 00:12:57,040 in class, even though we've only gone through seven lectures. 265 00:12:57,040 --> 00:13:01,030 And when we look at the LDL receptor and cholesterol 266 00:13:01,030 --> 00:13:04,890 homeostasis, there aren't very many LDL receptors. 267 00:13:04,890 --> 00:13:07,267 You something highly sensitive, which is something 268 00:13:07,267 --> 00:13:08,475 that you need to think about. 269 00:13:08,475 --> 00:13:11,080 And I-125 which is a gamma emitter, 270 00:13:11,080 --> 00:13:12,730 is what you end up using. 271 00:13:12,730 --> 00:13:14,590 We'll come back to that in recitation, 272 00:13:14,590 --> 00:13:16,770 I don't know, 8 I think it is. 273 00:13:16,770 --> 00:13:20,070 OK, so they're unstable. 274 00:13:20,070 --> 00:13:24,560 And they spontaneously decay into a new configuration. 275 00:13:24,560 --> 00:13:26,230 And they release energy. 276 00:13:26,230 --> 00:13:30,970 And what we want to do is detect the energy. 277 00:13:30,970 --> 00:13:34,990 The ones that most of you will be focused on, 278 00:13:34,990 --> 00:13:38,650 if you use radioactivity in your experiments, 279 00:13:38,650 --> 00:13:41,530 will probably be all beta emitters. 280 00:13:41,530 --> 00:13:45,190 And all of these guys over here are beta emitters. 281 00:13:45,190 --> 00:13:48,770 And you've already seen all of these radioisotopes. 282 00:13:48,770 --> 00:13:54,670 OK, so what do we know about these isotopes? 283 00:13:54,670 --> 00:13:56,990 There's two things you need to think about. 284 00:13:56,990 --> 00:14:00,000 So this is the properties. 285 00:14:00,000 --> 00:14:04,930 And one of them is the energy of the beta particle or gamma 286 00:14:04,930 --> 00:14:07,520 particle released. 287 00:14:07,520 --> 00:14:11,050 And if you look over here, what do you see? 288 00:14:11,050 --> 00:14:17,240 Tritium has 18.6 with this kind of unit. 289 00:14:17,240 --> 00:14:19,000 The unit might not mean very much to you. 290 00:14:19,000 --> 00:14:22,270 All I want you to do is look at the relative energies. 291 00:14:22,270 --> 00:14:27,850 Versus phosphorus, 1,710, so it has much more energy released. 292 00:14:27,850 --> 00:14:29,020 And what does that mean? 293 00:14:29,020 --> 00:14:31,540 If you've never worked with radioactivity, you might not-- 294 00:14:31,540 --> 00:14:34,870 a lot of chemists are petrified of radioactivity. 295 00:14:34,870 --> 00:14:37,060 I mean you could eat most tritium and C-14. 296 00:14:37,060 --> 00:14:38,650 Don't tell anybody I said that. 297 00:14:38,650 --> 00:14:41,440 But you could eat almost all tritium or C-14-labeled 298 00:14:41,440 --> 00:14:43,680 molecules you end up buying. 299 00:14:43,680 --> 00:14:47,740 They don't really do anything to you because the energy is low. 300 00:14:47,740 --> 00:14:50,080 And if you wear plastic gloves or something, 301 00:14:50,080 --> 00:14:53,670 that protects you from any kind of energy released. 302 00:14:53,670 --> 00:14:58,270 P-32, on the other hand, which isn't used as frequently, 303 00:14:58,270 --> 00:15:01,060 does anybody know where that used to be use all the time? 304 00:15:01,060 --> 00:15:04,710 I've used tons of P-32 in my lifetime. 305 00:15:04,710 --> 00:15:07,540 Where do you think that was used initially? 306 00:15:07,540 --> 00:15:08,622 AUDIENCE: With DNA. 307 00:15:08,622 --> 00:15:09,580 JOANNE STUBBE: In what? 308 00:15:09,580 --> 00:15:10,360 AUDIENCE: DNA. 309 00:15:10,360 --> 00:15:12,370 JOANNE STUBBE: DNA sequencing, yeah. 310 00:15:12,370 --> 00:15:14,770 So DNA sequencing, which you guys don't do, 311 00:15:14,770 --> 00:15:16,900 you send it out to have somebody do it for you. 312 00:15:16,900 --> 00:15:18,490 We used to run these huge gels. 313 00:15:18,490 --> 00:15:20,890 And we used to have to run many, many sequencing 314 00:15:20,890 --> 00:15:24,400 gels to sequence something that was 500 base pairs long. 315 00:15:24,400 --> 00:15:26,920 And P-32 was the method of detection. 316 00:15:26,920 --> 00:15:29,290 So what you do there is it's still not that bad, 317 00:15:29,290 --> 00:15:31,720 but you have to have a safety shield. 318 00:15:31,720 --> 00:15:34,900 So if any of you use radioactivity at MIT, 319 00:15:34,900 --> 00:15:37,287 they have radiation safety, and you go. 320 00:15:37,287 --> 00:15:39,370 Even if there's somebody else in the lab using it, 321 00:15:39,370 --> 00:15:43,090 and you're not, you should go just read the handouts 322 00:15:43,090 --> 00:15:44,950 that they give you to be aware of what's 323 00:15:44,950 --> 00:15:46,600 going on with radioactivity. 324 00:15:46,600 --> 00:15:50,050 I would say the biggest issue is that, if somebody 325 00:15:50,050 --> 00:15:52,810 spills it and doesn't clean it up, 326 00:15:52,810 --> 00:15:55,540 then it can contaminate everybody's experiments 327 00:15:55,540 --> 00:15:56,385 in the lab. 328 00:15:56,385 --> 00:15:58,060 That I would say is one of the biggest 329 00:15:58,060 --> 00:16:00,000 issues with radioactivity. 330 00:16:00,000 --> 00:16:05,260 OK, so iodine, again, is a gamma emitter. 331 00:16:05,260 --> 00:16:07,005 So that's in a category by itself. 332 00:16:07,005 --> 00:16:09,130 So the other thing that I think people don't really 333 00:16:09,130 --> 00:16:13,530 have very much feeling for is the half-life of decay. 334 00:16:13,530 --> 00:16:18,490 And if you look at that, look at how many years for your C-14 335 00:16:18,490 --> 00:16:23,246 to decay by 50% from whatever the number is, forever. 336 00:16:23,246 --> 00:16:24,620 You don't have to worry about it. 337 00:16:24,620 --> 00:16:25,840 You can sit it in your-- 338 00:16:25,840 --> 00:16:27,423 you can leave it in your refrigerator, 339 00:16:27,423 --> 00:16:29,560 and it's good for your lifetime anyhow. 340 00:16:29,560 --> 00:16:32,330 On the other hand, P-32, for example, 341 00:16:32,330 --> 00:16:34,510 has a half-life of 14 days. 342 00:16:34,510 --> 00:16:35,530 So what does that mean? 343 00:16:35,530 --> 00:16:39,040 It's spontaneously decaying continuously. 344 00:16:39,040 --> 00:16:43,120 And if you have it for 14 days, you start out with some number. 345 00:16:43,120 --> 00:16:45,200 We'll define what that number is. 346 00:16:45,200 --> 00:16:48,616 And then 14 days later, you only have half as much. 347 00:16:48,616 --> 00:16:51,340 OK, so you need to know something about the half-life. 348 00:16:51,340 --> 00:16:53,650 And the only one you need to ever think about 349 00:16:53,650 --> 00:16:55,840 is P-32, which they needed to think 350 00:16:55,840 --> 00:17:00,610 about in the experiments that are described in the Rodnina 351 00:17:00,610 --> 00:17:02,380 paper. 352 00:17:02,380 --> 00:17:07,170 So you have the energy released, and the energies are distinct. 353 00:17:07,170 --> 00:17:10,750 And so the question then is what we really want to do 354 00:17:10,750 --> 00:17:12,579 is think about quantitation. 355 00:17:12,579 --> 00:17:15,030 So that's going to be the key thing 356 00:17:15,030 --> 00:17:22,390 is we want to be able to quantitate radioactivity. 357 00:17:22,390 --> 00:17:26,319 And to do that, we need a method of detection. 358 00:17:29,610 --> 00:17:34,150 And there are a number of methods of detection. 359 00:17:34,150 --> 00:17:36,440 The one that-- 360 00:17:36,440 --> 00:17:38,254 I guess, again, I'm not sure. 361 00:17:38,254 --> 00:17:40,420 I think I'm the only one in the chemistry department 362 00:17:40,420 --> 00:17:43,750 that has a way of detecting radioactivity 363 00:17:43,750 --> 00:17:46,750 using an instrument called a scintillation counter. 364 00:17:46,750 --> 00:17:52,240 So this is sort of a very oversimplified view 365 00:17:52,240 --> 00:17:55,105 of what's going on in your scintillation counter. 366 00:17:55,105 --> 00:17:56,980 And so people come from all-- actually, 367 00:17:56,980 --> 00:17:59,330 they come from lots of places on campus to use it. 368 00:17:59,330 --> 00:18:00,790 So again, I think that's common. 369 00:18:00,790 --> 00:18:03,220 I don't know how many people are using radioactivity. 370 00:18:03,220 --> 00:18:05,240 But you have radiolabeled molecule here. 371 00:18:05,240 --> 00:18:07,240 It would be leucine. 372 00:18:07,240 --> 00:18:09,210 And you have tritiated leucine. 373 00:18:09,210 --> 00:18:13,210 OK, or it would be P-32-labelled GTP. 374 00:18:13,210 --> 00:18:17,980 Those are the two molecules that are used in these experiments. 375 00:18:17,980 --> 00:18:22,190 You put it into a little vessel with some kind of fluid. 376 00:18:22,190 --> 00:18:26,440 And the fluid that you use, whether it's organic, water, 377 00:18:26,440 --> 00:18:30,280 aqueous, or mixtures, depends on the molecules 378 00:18:30,280 --> 00:18:31,190 you're dealing with. 379 00:18:31,190 --> 00:18:35,500 OK, and the energy gets transferred in some way 380 00:18:35,500 --> 00:18:39,390 to the solvent in your solution. 381 00:18:39,390 --> 00:18:41,470 And then you put in a small molecule 382 00:18:41,470 --> 00:18:43,930 called the scintillant, which can 383 00:18:43,930 --> 00:18:46,920 remove the energy from the solvent 384 00:18:46,920 --> 00:18:50,310 and absorb that energy in some way. 385 00:18:50,310 --> 00:18:52,330 And again, it depends on-- the standard one 386 00:18:52,330 --> 00:18:54,190 we use in my lab is POPOP. 387 00:18:54,190 --> 00:18:56,680 You can look up scintillants in Google, 388 00:18:56,680 --> 00:18:59,930 and you can find out what the structures of these things are. 389 00:18:59,930 --> 00:19:01,840 And then these things decay. 390 00:19:01,840 --> 00:19:06,340 And when they decay, the energy is 391 00:19:06,340 --> 00:19:10,780 related to the detection method using a photomultiplier tube. 392 00:19:10,780 --> 00:19:13,360 So it gives you a quantitative measure 393 00:19:13,360 --> 00:19:18,640 of how much radioactivity you have over here 394 00:19:18,640 --> 00:19:22,330 and how much you get out on this side. 395 00:19:22,330 --> 00:19:23,830 Now, if you look at this process, 396 00:19:23,830 --> 00:19:26,890 it's complicated because you have energy transfer. 397 00:19:26,890 --> 00:19:30,160 So what can happen during this energy transfer 398 00:19:30,160 --> 00:19:33,760 depending on the energy of your radioisotope? 399 00:19:33,760 --> 00:19:35,410 Anybody got any ideas? 400 00:19:35,410 --> 00:19:37,820 What might you have to worry about? 401 00:19:37,820 --> 00:19:39,780 AUDIENCE: You're looking at efficient transfer. 402 00:19:39,780 --> 00:19:42,984 JOANNE STUBBE: Yeah, so the efficiency of the transfer. 403 00:19:42,984 --> 00:19:44,650 And if you have something in a solution, 404 00:19:44,650 --> 00:19:47,120 often, you're doing crude cell extracts. 405 00:19:47,120 --> 00:19:49,570 OK, so you have a lot of things in there 406 00:19:49,570 --> 00:19:52,030 that can also absorb the energy. 407 00:19:52,030 --> 00:19:55,844 So at any stage along the way, you can get quenching. 408 00:19:55,844 --> 00:19:59,710 OK, and if you get quenching, that reduces the amount you 409 00:19:59,710 --> 00:20:01,550 detect over here. 410 00:20:01,550 --> 00:20:04,480 OK, so that's going to throw your numbers off. 411 00:20:04,480 --> 00:20:06,610 So where is quenching a problem? 412 00:20:06,610 --> 00:20:08,560 Quenching is a problem-- 413 00:20:08,560 --> 00:20:10,640 we just looked at all these energies. 414 00:20:10,640 --> 00:20:14,480 OK, tritium has the lowest energy. 415 00:20:14,480 --> 00:20:18,740 OK, P-32 has a much higher energy. 416 00:20:18,740 --> 00:20:21,850 So if you look at it, and you have to figure this out 417 00:20:21,850 --> 00:20:23,770 for every system you work on. 418 00:20:23,770 --> 00:20:26,440 I've worked on tritium-labeled molecules 419 00:20:26,440 --> 00:20:29,720 where you couldn't be quenched by 90%. 420 00:20:29,720 --> 00:20:32,620 So if you have some measure-- we'll call it decompositions 421 00:20:32,620 --> 00:20:35,740 per minute, 10,000-- if it's quenched by 90%, 422 00:20:35,740 --> 00:20:39,040 you've lost a lot of your sensitivity. 423 00:20:39,040 --> 00:20:42,010 So you have to figure out a way to determine whether you'll 424 00:20:42,010 --> 00:20:43,000 get quenching or not. 425 00:20:43,000 --> 00:20:45,440 Otherwise, your numbers are completely off. 426 00:20:45,440 --> 00:20:50,470 So why do you want to quantitate your radioactivity? 427 00:20:50,470 --> 00:20:53,060 Where would you be using radioactivity? 428 00:20:53,060 --> 00:20:55,540 And why would this quenching make a difference? 429 00:20:55,540 --> 00:20:56,344 Yeah? 430 00:20:56,344 --> 00:20:57,760 AUDIENCE: This is just a question. 431 00:20:57,760 --> 00:20:59,113 JOANNE STUBBE: Yeah, sure. 432 00:20:59,113 --> 00:21:01,084 AUDIENCE: What's kind of like the nature 433 00:21:01,084 --> 00:21:04,130 of the solvent to the fluorescence involved. 434 00:21:04,130 --> 00:21:06,830 Is that like a [INAUDIBLE] kind of idea? 435 00:21:06,830 --> 00:21:10,530 JOANNE STUBBE: So it's just some kind of energy transfer. 436 00:21:10,530 --> 00:21:13,180 Yeah, so I mean it depends on what the molecules are. 437 00:21:13,180 --> 00:21:16,589 An it depends on what the solvent is. 438 00:21:16,589 --> 00:21:17,130 AUDIENCE: OK. 439 00:21:17,130 --> 00:21:22,400 JOANNE STUBBE: OK, so every single one of these systems, 440 00:21:22,400 --> 00:21:25,460 you need to go in and look at the details of what's going on. 441 00:21:25,460 --> 00:21:27,880 And so when you do this, people have worked out 442 00:21:27,880 --> 00:21:31,200 these conditions so that when you're measuring-- 443 00:21:31,200 --> 00:21:33,909 and so this is an important question you're asking. 444 00:21:33,909 --> 00:21:35,950 How do you know that what you're measuring really 445 00:21:35,950 --> 00:21:38,540 is related to what's way over here? 446 00:21:38,540 --> 00:21:42,684 So that's the absolutely the right question to ask. 447 00:21:42,684 --> 00:21:45,100 And so when you start, for example, the first thing you do 448 00:21:45,100 --> 00:21:48,490 is every scintillation counter comes with a standard. 449 00:21:48,490 --> 00:21:51,220 OK, and so the instrument is calibrated. 450 00:21:51,220 --> 00:21:53,251 And if you care about radioactivity, 451 00:21:53,251 --> 00:21:55,750 you have somebody come in, and they calibrate the instrument 452 00:21:55,750 --> 00:21:56,845 twice a year. 453 00:21:56,845 --> 00:22:00,520 OK, so all of this stuff is really important. 454 00:22:00,520 --> 00:22:02,850 And the question of sensitivity is important. 455 00:22:02,850 --> 00:22:05,350 We're going to see what you're measuring is something called 456 00:22:05,350 --> 00:22:07,200 decompositions per minute. 457 00:22:07,200 --> 00:22:11,180 OK, that's the readout you get from the instrument. 458 00:22:11,180 --> 00:22:15,740 And so you might be getting 100,000 of these things. 459 00:22:15,740 --> 00:22:18,082 But in fact, you might be getting five. 460 00:22:18,082 --> 00:22:20,360 OK, is five real? 461 00:22:20,360 --> 00:22:24,310 Five can be real if you count it so you 462 00:22:24,310 --> 00:22:26,740 get a statistical distribution to make 463 00:22:26,740 --> 00:22:30,130 sure the five is real, that it's not five plus or minus five. 464 00:22:30,130 --> 00:22:34,390 OK, so radioactivity is incredibly sensitive. 465 00:22:34,390 --> 00:22:36,040 And you can extend the sensitivity 466 00:22:36,040 --> 00:22:39,910 by just counting your material in this instrument 467 00:22:39,910 --> 00:22:42,930 for a very long period of time. 468 00:22:42,930 --> 00:22:45,550 OK, so where would you want-- 469 00:22:45,550 --> 00:22:50,820 where have you already seen that you would use radioisotopes? 470 00:22:50,820 --> 00:22:57,070 What did you see today in class, for example, in ribo-x? 471 00:22:57,070 --> 00:23:00,130 You looked at an experiment with ribo-x today in class. 472 00:23:00,130 --> 00:23:02,026 AUDIENCE: Oh, the cysteine incorporation. 473 00:23:02,026 --> 00:23:04,067 JOANNE STUBBE: Yeah, with cysteine incorporation. 474 00:23:04,067 --> 00:23:06,940 What were you looking at? 475 00:23:06,940 --> 00:23:09,330 AUDIENCE: It limited the radioactive system. 476 00:23:09,330 --> 00:23:11,100 JOANNE STUBBE: Right, but what we are using to look at this? 477 00:23:11,100 --> 00:23:13,060 So we're talking about the detection method. 478 00:23:13,060 --> 00:23:15,590 So I'm going to describe another detection method. 479 00:23:15,590 --> 00:23:18,200 Would you be looking at this by scintillation counting? 480 00:23:18,200 --> 00:23:20,510 No, so you need another method. 481 00:23:20,510 --> 00:23:22,812 AUDIENCE: So like one of those phosphorimagers. 482 00:23:22,812 --> 00:23:24,770 JOANNE STUBBE: Right, so I'm going to show you. 483 00:23:24,770 --> 00:23:25,686 That's the next thing. 484 00:23:25,686 --> 00:23:29,490 So what you could have, for example, is a TLC plate. 485 00:23:29,490 --> 00:23:30,660 Or you could have-- 486 00:23:30,660 --> 00:23:34,400 they were using a gel, an agarose gel probably. 487 00:23:34,400 --> 00:23:37,200 So you need a way of detect the radioactivity that's 488 00:23:37,200 --> 00:23:39,200 going to be distinct from scintillation counters 489 00:23:39,200 --> 00:23:43,290 where you use little vials and scintillation fluid. 490 00:23:43,290 --> 00:23:46,760 And you have a completely different method of detection. 491 00:23:46,760 --> 00:23:49,470 And these methods keep changing. 492 00:23:49,470 --> 00:23:52,730 And so I don't update them anymore. 493 00:23:52,730 --> 00:23:54,740 I'm not sure what the current technology is. 494 00:23:54,740 --> 00:23:57,780 It's all secret anyhow. 495 00:23:57,780 --> 00:23:59,951 So they tell you sort of something about what it is, 496 00:23:59,951 --> 00:24:01,700 but they don't tell you any of the details 497 00:24:01,700 --> 00:24:04,270 because it's all proprietary. 498 00:24:04,270 --> 00:24:08,960 OK, so in that case, what were we looking at? 499 00:24:08,960 --> 00:24:10,970 We were just looking for incorporation. 500 00:24:10,970 --> 00:24:13,900 We were doing some labeling experiment in the cell. 501 00:24:13,900 --> 00:24:16,040 OK, so we were chasing a label. 502 00:24:16,040 --> 00:24:18,150 So that you're going to see a lot. 503 00:24:20,720 --> 00:24:24,320 That's how all the metabolic pathways were figured out. 504 00:24:24,320 --> 00:24:28,160 The advent of C-14 as an isotopic label 505 00:24:28,160 --> 00:24:32,180 revolutionized our understanding of glycolysis, fatty 506 00:24:32,180 --> 00:24:35,180 acid biosynthesis, et cetera. 507 00:24:35,180 --> 00:24:40,610 And today, what are we using radioisotopes today to do? 508 00:24:40,610 --> 00:24:43,490 We're using it to do what? 509 00:24:43,490 --> 00:24:44,525 We're looking at GTP. 510 00:24:47,630 --> 00:24:53,816 We want to look at GTP going to GDP plus Pi. 511 00:24:53,816 --> 00:24:57,170 OK, so what are we using this for? 512 00:24:57,170 --> 00:25:01,100 To get information for our model. 513 00:25:01,100 --> 00:25:02,690 What do we do as a function of time? 514 00:25:06,910 --> 00:25:12,180 Why do we want to use gamma P-32-labeled ATP? 515 00:25:12,180 --> 00:25:16,100 And how do we use this in analysis? 516 00:25:16,100 --> 00:25:20,310 Did you even know that we're using gamma P-32-labeled GDP? 517 00:25:20,310 --> 00:25:22,740 How many knew that? 518 00:25:22,740 --> 00:25:24,915 Anybody read that in the methods section? 519 00:25:27,970 --> 00:25:32,252 You, over there, did you read that in the methods section? 520 00:25:32,252 --> 00:25:32,960 What's your name? 521 00:25:32,960 --> 00:25:33,970 AUDIENCE: Mathis. 522 00:25:33,970 --> 00:25:34,440 JOANNE STUBBE: Matt? 523 00:25:34,440 --> 00:25:35,200 AUDIENCE: Mathis. 524 00:25:35,200 --> 00:25:37,570 JOANNE STUBBE: Matt, did you read that in the methods 525 00:25:37,570 --> 00:25:38,770 section or not? 526 00:25:38,770 --> 00:25:39,650 AUDIENCE: No. 527 00:25:39,650 --> 00:25:43,050 JOANNE STUBBE: No, did anybody read it in the methods section? 528 00:25:43,050 --> 00:25:44,750 So I mean that's what-- again, this 529 00:25:44,750 --> 00:25:48,320 is what this recitation is all about 530 00:25:48,320 --> 00:25:51,830 is looking at the details of what's going on. 531 00:25:51,830 --> 00:25:54,540 And I think when you first start doing something like you don't 532 00:25:54,540 --> 00:25:56,030 know what details to look for. 533 00:25:56,030 --> 00:25:59,220 Some of you might have read it, but it didn't mean anything. 534 00:25:59,220 --> 00:26:00,921 So it went in one ear and out the other. 535 00:26:00,921 --> 00:26:01,420 Yeah? 536 00:26:01,420 --> 00:26:04,930 AUDIENCE: Wouldn't you have to use gamma labeled GTP though? 537 00:26:04,930 --> 00:26:09,622 I mean the hydrolysis gives you GDP and phosphate. 538 00:26:09,622 --> 00:26:11,882 So that's the only-- 539 00:26:11,882 --> 00:26:13,340 I mean, if you labeled another one, 540 00:26:13,340 --> 00:26:15,006 it doesn't give you as much information. 541 00:26:15,006 --> 00:26:17,660 JOANNE STUBBE: OK, so if you labeled-- 542 00:26:17,660 --> 00:26:20,340 say you labeled the base. 543 00:26:20,340 --> 00:26:22,760 So let's just call this base. 544 00:26:22,760 --> 00:26:33,940 Say you put a tritium in the base, OK, versus-- 545 00:26:33,940 --> 00:26:36,820 hopefully, you all know this, but this is the gamma position 546 00:26:36,820 --> 00:26:38,110 versus a label here. 547 00:26:38,110 --> 00:26:39,580 Why are you putting the label here? 548 00:26:43,730 --> 00:26:45,539 What's going on in this reaction? 549 00:26:45,539 --> 00:26:47,330 Actually, this was interesting because this 550 00:26:47,330 --> 00:26:49,955 is the second recitation where I don't think anybody understood 551 00:26:49,955 --> 00:26:52,640 what was going on in this reaction, which 552 00:26:52,640 --> 00:26:54,080 is rather disturbing. 553 00:26:54,080 --> 00:26:55,980 What's going on in this reaction? 554 00:26:55,980 --> 00:26:57,560 So we're going GTP. 555 00:26:57,560 --> 00:27:01,010 So this is G. So you have a nucleoside 556 00:27:01,010 --> 00:27:03,340 and three phosphates, TP. 557 00:27:03,340 --> 00:27:06,110 And what are you producing out the other side? 558 00:27:06,110 --> 00:27:07,280 GDP. 559 00:27:07,280 --> 00:27:11,200 So what's happening during this reaction? 560 00:27:11,200 --> 00:27:11,856 Yeah? 561 00:27:11,856 --> 00:27:13,480 AUDIENCE: [INAUDIBLE] 562 00:27:13,480 --> 00:27:15,560 JOANNE STUBBE: Yeah, so you're hydrolyzing it. 563 00:27:15,560 --> 00:27:20,640 So in some way, that's what all of these GTPases are about. 564 00:27:20,640 --> 00:27:23,150 You're going to see these GTPases not only 565 00:27:23,150 --> 00:27:24,020 in translation. 566 00:27:24,020 --> 00:27:26,180 You're going to see it in three of the sections 567 00:27:26,180 --> 00:27:26,930 that I talk about. 568 00:27:26,930 --> 00:27:29,140 GTPases are everywhere. 569 00:27:29,140 --> 00:27:32,240 OK, so what you're looking at is then 570 00:27:32,240 --> 00:27:36,590 some way you have hydrolysis of the gamma phosphate. 571 00:27:36,590 --> 00:27:40,280 OK, so why are you labeling the gamma phosphate? 572 00:27:40,280 --> 00:27:43,700 You could have labeled actually the alpha or the beta. 573 00:27:46,430 --> 00:27:48,430 AUDIENCE: You wouldn't be watching the reacting. 574 00:27:48,430 --> 00:27:50,670 JOANNE STUBBE: Yeah, you want to watch your reaction. 575 00:27:50,670 --> 00:27:53,060 So if you have an isotope here, which 576 00:27:53,060 --> 00:27:54,910 we're going to watch it using some method, 577 00:27:54,910 --> 00:27:58,410 scintillation counting or phosphorimaging, and where 578 00:27:58,410 --> 00:28:01,580 does the label end up? 579 00:28:01,580 --> 00:28:03,870 The label ends up here. 580 00:28:03,870 --> 00:28:07,545 OK, well, if you put the label in alpha or beta, 581 00:28:07,545 --> 00:28:08,795 could you follow the reaction? 582 00:28:14,000 --> 00:28:16,000 OK, well, you're shaking your head no. 583 00:28:16,000 --> 00:28:17,807 Why couldn't you follow the reaction? 584 00:28:17,807 --> 00:28:20,265 AUDIENCE: Because it would stick in the GDP [INAUDIBLE]---- 585 00:28:20,265 --> 00:28:21,806 JOANNE STUBBE: So it would be in GDP. 586 00:28:21,806 --> 00:28:23,356 AUDIENCE: --there's no GTP in GDP. 587 00:28:23,356 --> 00:28:25,106 JOANNE STUBBE: Yeah, is there a difference 588 00:28:25,106 --> 00:28:26,665 chemically between GDP and GTP? 589 00:28:30,130 --> 00:28:31,900 Again, this is what I'm finding. 590 00:28:31,900 --> 00:28:35,680 You need to think about the structures of everything 591 00:28:35,680 --> 00:28:36,520 you're working with. 592 00:28:36,520 --> 00:28:37,530 We're chemists. 593 00:28:37,530 --> 00:28:40,540 OK, what is the difference between the diphosphate 594 00:28:40,540 --> 00:28:43,762 and the triphosphate? 595 00:28:43,762 --> 00:28:45,970 AUDIENCE: It's harder to hydrolyze the next phosphate 596 00:28:45,970 --> 00:28:46,680 off. 597 00:28:46,680 --> 00:28:48,370 JOANNE STUBBE: Well, it's not. 598 00:28:48,370 --> 00:28:51,080 You know, all of these things-- 599 00:28:51,080 --> 00:28:53,700 without an enzyme, all of these things are hard to hydrolyze. 600 00:28:53,700 --> 00:28:54,200 Why? 601 00:28:54,200 --> 00:28:56,660 Because you've got negative charges all over the place, 602 00:28:56,660 --> 00:28:59,126 and a nucleophile can't get into the active site. 603 00:28:59,126 --> 00:29:00,500 So they're all hard to hydrolyze. 604 00:29:00,500 --> 00:29:03,260 So that's not-- you have to think about that, 605 00:29:03,260 --> 00:29:05,100 but that's not what I'm looking for. 606 00:29:05,100 --> 00:29:05,790 Yeah? 607 00:29:05,790 --> 00:29:08,050 AUDIENCE: So if you run a gel or something, 608 00:29:08,050 --> 00:29:09,140 they should come out-- 609 00:29:09,140 --> 00:29:13,346 GDP and GTP are going to come out in the same-ish area, 610 00:29:13,346 --> 00:29:14,730 whereas, obviously, phosphate-- 611 00:29:14,730 --> 00:29:17,146 JOANNE STUBBE: OK, so that's what you need to think about. 612 00:29:17,146 --> 00:29:19,910 But what can you take advantage of as a chemist where 613 00:29:19,910 --> 00:29:23,211 they don't come out in the same-ish area? 614 00:29:23,211 --> 00:29:24,710 AUDIENCE: I mean, if you label them, 615 00:29:24,710 --> 00:29:27,238 the gamma phosphate, then the label 616 00:29:27,238 --> 00:29:29,180 won't come out nearly anywhere. 617 00:29:29,180 --> 00:29:31,090 JOANNE STUBBE: So that's absolutely true. 618 00:29:31,090 --> 00:29:33,450 But what is it about this molecule? 619 00:29:33,450 --> 00:29:34,770 Because I've been sloppy. 620 00:29:34,770 --> 00:29:37,380 What is it about this molecule that 621 00:29:37,380 --> 00:29:41,114 allows the distinction between your starting materials 622 00:29:41,114 --> 00:29:41,655 and products? 623 00:29:41,655 --> 00:29:44,460 This is what developing an assay is all about. 624 00:29:44,460 --> 00:29:47,240 How are you going to monitor this reaction? 625 00:29:47,240 --> 00:29:52,200 So in this paper, one of the graphs looked at Pi production. 626 00:29:52,200 --> 00:29:54,720 We're going to look at this if we get this far. 627 00:29:54,720 --> 00:29:57,509 OK, so how would you distinguish between these things 628 00:29:57,509 --> 00:29:58,050 as a chemist? 629 00:30:02,940 --> 00:30:05,990 You have no idea. 630 00:30:05,990 --> 00:30:09,590 You, you haven't any idea, not good. 631 00:30:09,590 --> 00:30:12,020 OK, what about you? 632 00:30:12,020 --> 00:30:14,150 This isn't a hard question. 633 00:30:14,150 --> 00:30:15,870 Look at the structures. 634 00:30:15,870 --> 00:30:18,410 And as a chemist, how would you distinguish 635 00:30:18,410 --> 00:30:20,200 your starting material from your products? 636 00:30:20,200 --> 00:30:21,320 That's the question. 637 00:30:21,320 --> 00:30:25,750 And that is the question in any assay you have to develop. 638 00:30:25,750 --> 00:30:27,680 That's what you've got to figure out. 639 00:30:27,680 --> 00:30:30,590 You've got to figure out a way to distinguish 640 00:30:30,590 --> 00:30:32,330 the starting materials from the product. 641 00:30:32,330 --> 00:30:37,040 Now, if we have a base here, and if this is G, 642 00:30:37,040 --> 00:30:38,030 we have a base here. 643 00:30:38,030 --> 00:30:41,360 What do we know about guanine? 644 00:30:41,360 --> 00:30:42,980 What's its absorption look like? 645 00:30:42,980 --> 00:30:45,752 What's its absorption spectrum look like? 646 00:30:45,752 --> 00:30:47,480 AUDIENCE: 210 [INAUDIBLE]. 647 00:30:47,480 --> 00:30:49,421 JOANNE STUBBE: How much? 648 00:30:49,421 --> 00:30:51,987 AUDIENCE: Isn't it like 210 nanometers. 649 00:30:51,987 --> 00:30:53,320 JOANNE STUBBE: I can't hear you. 650 00:30:53,320 --> 00:30:54,652 You need to-- don't mumble. 651 00:30:54,652 --> 00:30:56,110 Look at me in the face and tell me. 652 00:30:56,110 --> 00:30:57,770 You know, don't be shy. 653 00:30:57,770 --> 00:30:59,690 I mean, we all ask questions. 654 00:30:59,690 --> 00:31:00,190 [INAUDIBLE] 655 00:31:00,190 --> 00:31:01,150 We're here to learn. 656 00:31:01,150 --> 00:31:01,900 Right? 657 00:31:01,900 --> 00:31:02,110 Yeah? 658 00:31:02,110 --> 00:31:02,980 AUDIENCE: It absorbs in the UV. 659 00:31:02,980 --> 00:31:04,480 I think it's 210 nanometers. 660 00:31:04,480 --> 00:31:06,190 JOANNE STUBBE: OK, so it's not 210. 661 00:31:06,190 --> 00:31:09,250 So you guys need to go think about amino acids 662 00:31:09,250 --> 00:31:10,060 and nucleic acid. 663 00:31:10,060 --> 00:31:12,160 It absorbs at 260. 664 00:31:12,160 --> 00:31:16,360 OK, so I mean, potentially, you could 665 00:31:16,360 --> 00:31:19,090 sit at this absorption at 260. 666 00:31:19,090 --> 00:31:21,730 But what does GTP look like? 667 00:31:21,730 --> 00:31:23,860 GDP look like? 668 00:31:23,860 --> 00:31:25,427 It has the same base. 669 00:31:25,427 --> 00:31:27,010 So you're not going to see any change. 670 00:31:27,010 --> 00:31:28,930 So that's useless because you need 671 00:31:28,930 --> 00:31:32,510 to be able to monitor a change during the reaction. 672 00:31:32,510 --> 00:31:35,800 OK, so what else about this molecule 673 00:31:35,800 --> 00:31:38,440 will easily let you, as a chemist, 674 00:31:38,440 --> 00:31:43,035 determine substrate from product? 675 00:31:43,035 --> 00:31:43,910 AUDIENCE: The charge. 676 00:31:43,910 --> 00:31:46,150 JOANNE STUBBE: Yeah, the charge, yes. 677 00:31:46,150 --> 00:31:48,010 AUDIENCE: Just do anything with the charge. 678 00:31:48,010 --> 00:31:50,320 JOANNE STUBBE: So here we have all of these negative-- 679 00:31:50,320 --> 00:31:52,126 every oxygen is negatively charged. 680 00:31:52,126 --> 00:31:53,500 Here we only have two phosphates. 681 00:31:53,500 --> 00:31:55,510 Every oxygen is negatively charged. 682 00:31:55,510 --> 00:31:58,000 Phosphate-- all right, let me ask this question. 683 00:31:58,000 --> 00:32:01,360 We'll see how much we need to be thinking about here. 684 00:32:01,360 --> 00:32:05,980 So we have-- what is the charged state of phosphate? 685 00:32:05,980 --> 00:32:07,030 Can anybody tell me? 686 00:32:10,862 --> 00:32:11,820 AUDIENCE: Minus 3. 687 00:32:11,820 --> 00:32:12,861 JOANNE STUBBE: Pardon me? 688 00:32:12,861 --> 00:32:13,946 AUDIENCE: Minus 3. 689 00:32:13,946 --> 00:32:15,880 It depends on the pH of your solution. 690 00:32:15,880 --> 00:32:18,150 JOANNE STUBBE: Yeah, well, we're at neutral pH. 691 00:32:18,150 --> 00:32:19,610 So you look at all the buffer. 692 00:32:19,610 --> 00:32:21,820 You know what the buffers are. 693 00:32:21,820 --> 00:32:24,230 They've described the buffer in their reaction. 694 00:32:24,230 --> 00:32:25,490 So you're at neutral pH. 695 00:32:25,490 --> 00:32:27,671 What is the charge? 696 00:32:27,671 --> 00:32:29,459 AUDIENCE: Minus 2. 697 00:32:29,459 --> 00:32:34,560 JOANNE STUBBE: Yeah, so it's the pKa of the first proton loss 698 00:32:34,560 --> 00:32:35,670 is at 1.6. 699 00:32:35,670 --> 00:32:39,100 And the pKa of the second proton loss is about 6.8. 700 00:32:39,100 --> 00:32:42,870 So you'll have a mixture between 1 and 2. 701 00:32:42,870 --> 00:32:46,650 So this is incredibly different from this. 702 00:32:46,650 --> 00:32:48,720 And that makes it-- how do you separate things? 703 00:32:48,720 --> 00:32:51,420 By an anion exchange column, which separates things 704 00:32:51,420 --> 00:32:55,140 based on charge, some kind of a TLC system, 705 00:32:55,140 --> 00:32:58,540 which can separate things based on charge. 706 00:32:58,540 --> 00:33:03,562 And so that's what you have to do in your overall assay. 707 00:33:03,562 --> 00:33:06,750 OK, so the second place where you're 708 00:33:06,750 --> 00:33:10,180 going to use radioactivity is an assay. 709 00:33:10,180 --> 00:33:16,050 OK, and in the paper you read, not only did they 710 00:33:16,050 --> 00:33:22,080 use it for GTP, they had to use it to monitor peptide bond 711 00:33:22,080 --> 00:33:22,590 formation. 712 00:33:22,590 --> 00:33:25,750 Can anybody tell me how they did that? 713 00:33:25,750 --> 00:33:29,430 So what are we looking at if we go back to the original? 714 00:33:29,430 --> 00:33:34,020 What's the product of the reaction of the EF-Tu reaction 715 00:33:34,020 --> 00:33:35,464 with the ribosome? 716 00:33:40,410 --> 00:33:43,131 What's the product you get out? 717 00:33:43,131 --> 00:33:45,993 AUDIENCE: [INAUDIBLE] on EF-Tu and also 718 00:33:45,993 --> 00:33:49,359 label the hydrogen on leucine. 719 00:33:49,359 --> 00:33:50,900 JOANNE STUBBE: OK, so you're labeling 720 00:33:50,900 --> 00:33:52,220 the hydrogen on leucine. 721 00:33:52,220 --> 00:33:54,680 OK, but then what are you looking at in your assay? 722 00:33:54,680 --> 00:33:55,970 We're developing an assay. 723 00:33:55,970 --> 00:33:57,530 Here we're developing an assay where 724 00:33:57,530 --> 00:34:01,640 GTP is going to GDP plus Pi. 725 00:34:01,640 --> 00:34:04,760 What are we looking at in the case of the leucine 726 00:34:04,760 --> 00:34:05,799 in this experiment? 727 00:34:05,799 --> 00:34:08,090 AUDIENCE: The leucine is incorporated into the peptide. 728 00:34:08,090 --> 00:34:11,500 And you have the [INAUDIBLE]. 729 00:34:11,500 --> 00:34:14,820 JOANNE STUBBE: OK, so but where is the dipeptide? 730 00:34:14,820 --> 00:34:16,239 So that's correct, yeah. 731 00:34:16,239 --> 00:34:20,760 AUDIENCE: It will be in a P [INAUDIBLE] on the ribosome. 732 00:34:20,760 --> 00:34:23,360 JOANNE STUBBE: Yeah, but what's it attached to? 733 00:34:23,360 --> 00:34:24,409 Is it a dipeptide? 734 00:34:24,409 --> 00:34:27,489 AUDIENCE: Yeah, it's attached to the less phenylalanine. 735 00:34:27,489 --> 00:34:30,836 JOANNE STUBBE: Yeah, and what is that attached to? 736 00:34:30,836 --> 00:34:32,414 AUDIENCE: Another tRNA. 737 00:34:32,414 --> 00:34:34,580 JOANNE STUBBE: What's the phenylalanine attached to? 738 00:34:34,580 --> 00:34:37,550 If you look over here, what is everything attached to? 739 00:34:37,550 --> 00:34:38,510 AUDIENCE: Another tRNA. 740 00:34:38,510 --> 00:34:40,260 JOANNE STUBBE: It's attached to a tRNA. 741 00:34:40,260 --> 00:34:45,380 So could you separate a tRNA with one versus two amino acids 742 00:34:45,380 --> 00:34:45,889 chemically? 743 00:34:45,889 --> 00:34:48,658 Is that easy? 744 00:34:48,658 --> 00:34:49,199 AUDIENCE: No. 745 00:34:49,199 --> 00:34:50,960 JOANNE STUBBE: Now you have charges. 746 00:34:50,960 --> 00:34:51,460 Right? 747 00:34:51,460 --> 00:34:53,860 You have huge numbers of charges on your RNA. 748 00:34:53,860 --> 00:34:56,600 But they're the same on all the tRNAs. 749 00:34:56,600 --> 00:34:58,450 So you have one amino acid, which 750 00:34:58,450 --> 00:35:02,270 has a carboxylate end and a second amino acid, 751 00:35:02,270 --> 00:35:03,430 which has the same charge. 752 00:35:03,430 --> 00:35:05,471 Do you think that's going to be easy to separate? 753 00:35:05,471 --> 00:35:06,250 No. 754 00:35:06,250 --> 00:35:11,833 So does anybody know what they did to make this assay work? 755 00:35:11,833 --> 00:35:13,276 AUDIENCE: Put the label on leucine 756 00:35:13,276 --> 00:35:14,719 so the leucine is incorporated. 757 00:35:14,719 --> 00:35:17,124 Then you're still different [INAUDIBLE].. 758 00:35:17,124 --> 00:35:18,567 You can have basic number. 759 00:35:18,567 --> 00:35:21,264 Then after the conversion, you have a signal. 760 00:35:21,264 --> 00:35:23,680 JOANNE STUBBE: OK, so after conversion, you have a signal. 761 00:35:23,680 --> 00:35:26,440 But then the question is how do you detect this. 762 00:35:26,440 --> 00:35:27,139 So you have-- 763 00:35:27,139 --> 00:35:29,680 I mean, I guess what they could have done-- so we started out 764 00:35:29,680 --> 00:35:32,740 with a leucine that's labeled. 765 00:35:32,740 --> 00:35:34,960 And so what you're saying is that you 766 00:35:34,960 --> 00:35:41,800 have a way of detecting your leucine on the tRNA. 767 00:35:41,800 --> 00:35:46,280 So this is all attached through an ester linkage. 768 00:35:46,280 --> 00:35:48,080 So this is attached to the tRNA. 769 00:35:48,080 --> 00:35:51,160 So what you would be after is separating an amino acid 770 00:35:51,160 --> 00:35:52,300 from a tRNA. 771 00:35:52,300 --> 00:35:53,410 So that's possible. 772 00:35:53,410 --> 00:35:54,824 You could potentially do that. 773 00:35:54,824 --> 00:35:56,740 But what do you think about the ester linkage? 774 00:35:59,360 --> 00:36:02,450 This is all the thought process that goes into an assay 775 00:36:02,450 --> 00:36:04,220 and making an assay robust. 776 00:36:04,220 --> 00:36:07,680 Do you think that ester linkage is stable? 777 00:36:07,680 --> 00:36:09,680 You're going to have to chromatograph it someway 778 00:36:09,680 --> 00:36:13,700 to separate your starting material from product. 779 00:36:13,700 --> 00:36:16,100 So the answer is it's not very stable. 780 00:36:16,100 --> 00:36:18,450 And if you don't know, you've got to figure that out. 781 00:36:18,450 --> 00:36:26,132 So what they do is they quench the reaction with hydroxide. 782 00:36:26,132 --> 00:36:29,340 OK, and why did they quench the reaction with hydroxide? 783 00:36:29,340 --> 00:36:31,070 So this is a rapid chemical quench 784 00:36:31,070 --> 00:36:33,410 like we talked about last time. 785 00:36:33,410 --> 00:36:34,490 Why did they do that? 786 00:36:39,770 --> 00:36:41,690 AUDIENCE: To hydrolyze the ester. 787 00:36:41,690 --> 00:36:44,160 JOANNE STUBBE: Exactly, so then what do you have? 788 00:36:44,160 --> 00:36:47,660 You have, you know, your dipeptide here. 789 00:36:47,660 --> 00:36:50,970 Or you could hydrolyze before. 790 00:36:50,970 --> 00:36:53,410 And then you would have no label at all. 791 00:36:53,410 --> 00:36:56,070 And so then you can monitor dipeptide formation. 792 00:36:56,070 --> 00:36:59,270 So if you looked at the details of the graph 793 00:36:59,270 --> 00:37:00,920 that they presented, they weren't 794 00:37:00,920 --> 00:37:04,340 looking at tRNA charged with a dipeptide. 795 00:37:04,340 --> 00:37:06,905 They were looking at the peptide. 796 00:37:06,905 --> 00:37:08,810 And so that should have been a clue. 797 00:37:08,810 --> 00:37:10,760 Immediately, you go back to understand 798 00:37:10,760 --> 00:37:13,840 what's going on in the assay. 799 00:37:13,840 --> 00:37:15,740 So you have assays. 800 00:37:15,740 --> 00:37:16,910 This is pretty important. 801 00:37:16,910 --> 00:37:18,500 And where's another place where you 802 00:37:18,500 --> 00:37:21,350 might want to use radio label, where 803 00:37:21,350 --> 00:37:22,610 you need a sensitive assay? 804 00:37:22,610 --> 00:37:25,010 We're going to see radioactivity is incredibly sensitive. 805 00:37:25,010 --> 00:37:26,120 I'm not getting very far. 806 00:37:26,120 --> 00:37:29,600 But what other kind of an experiment 807 00:37:29,600 --> 00:37:31,760 might you think about if you have 808 00:37:31,760 --> 00:37:35,600 some kind of a mammalian cell, and you have receptors 809 00:37:35,600 --> 00:37:38,190 on the cell, for example? 810 00:37:38,190 --> 00:37:40,850 And you don't have very many receptors on the cell. 811 00:37:40,850 --> 00:37:44,510 You have, you know, sub-nanomolar number 812 00:37:44,510 --> 00:37:46,310 of receptors. 813 00:37:46,310 --> 00:37:48,770 Where else might you want to use radioactivity? 814 00:37:48,770 --> 00:37:52,430 And we're going to see this in the cholesterol section again. 815 00:37:52,430 --> 00:37:53,750 Anybody got any idea? 816 00:37:53,750 --> 00:37:55,250 So you have some receptor on a cell. 817 00:37:55,250 --> 00:37:58,440 And you want to count the number of receptors. 818 00:37:58,440 --> 00:38:01,550 We need a quantitative way of looking at that. 819 00:38:05,478 --> 00:38:07,100 AUDIENCE: We need to measure uptake. 820 00:38:07,100 --> 00:38:09,190 JOANNE STUBBE: So uptake is another place. 821 00:38:09,190 --> 00:38:12,010 You absolutely would want to use it to measure uptake. 822 00:38:12,010 --> 00:38:15,130 It's frequently used also to measure binding. 823 00:38:15,130 --> 00:38:19,530 OK, so you have to figure out a way to prevent-- 824 00:38:19,530 --> 00:38:22,720 on the cell, you can prevent uptake by just cooling down 825 00:38:22,720 --> 00:38:23,620 the lipids. 826 00:38:23,620 --> 00:38:25,350 And then you're measuring binding. 827 00:38:25,350 --> 00:38:28,410 OK, and that's exactly what they did in the LDL 828 00:38:28,410 --> 00:38:30,670 where they count the number of LDL receptors. 829 00:38:30,670 --> 00:38:32,560 So the other place where you're going 830 00:38:32,560 --> 00:38:35,180 to see this used over and over again 831 00:38:35,180 --> 00:38:37,099 is some kind of binding assay. 832 00:38:37,099 --> 00:38:38,890 And there are many ways to measure binding. 833 00:38:38,890 --> 00:38:40,990 You're going to have a whole recitation on this. 834 00:38:40,990 --> 00:38:47,010 Most of them aren't as sensitive as the radioactive methodology. 835 00:38:47,010 --> 00:38:54,160 OK, so let's move on after that long digression. 836 00:38:54,160 --> 00:38:57,250 OK, so what you need then is a quantitative way 837 00:38:57,250 --> 00:38:58,760 to measure radioactivity. 838 00:38:58,760 --> 00:39:02,344 OK, oh, the other thing I wanted to just point out, 839 00:39:02,344 --> 00:39:03,760 as you pointed out before, there's 840 00:39:03,760 --> 00:39:06,430 another way of detecting radioactivity 841 00:39:06,430 --> 00:39:08,110 using a phosphorimager. 842 00:39:08,110 --> 00:39:10,255 And you can read about this in detail. 843 00:39:10,255 --> 00:39:15,220 So what you do is you have your gel or a TLC plate. 844 00:39:15,220 --> 00:39:17,470 You have an image plate on top of it 845 00:39:17,470 --> 00:39:21,550 that somehow collects all the energy emitted 846 00:39:21,550 --> 00:39:23,470 from your radioactive decay. 847 00:39:23,470 --> 00:39:26,110 And then you quantitatively release that energy 848 00:39:26,110 --> 00:39:27,730 in a way that allows you to quantitate 849 00:39:27,730 --> 00:39:32,250 the amount of radioactivity you have 850 00:39:32,250 --> 00:39:36,560 on your spot on the gel or your spot on the TLC plate. 851 00:39:36,560 --> 00:39:39,920 And for example, tritium, with the lowest energy, 852 00:39:39,920 --> 00:39:43,570 you might have to put a plate onto your gel 853 00:39:43,570 --> 00:39:44,830 for a month and a half. 854 00:39:44,830 --> 00:39:46,855 That's how insensitive it is. 855 00:39:46,855 --> 00:39:49,480 You don't have enough energy to collect enough data to give you 856 00:39:49,480 --> 00:39:50,680 some kind of an answer. 857 00:39:50,680 --> 00:39:52,540 So you need to think about the energy, 858 00:39:52,540 --> 00:39:54,940 and you need to think about the method of detection. 859 00:39:54,940 --> 00:39:56,830 Tritium is the cheapest. 860 00:39:56,830 --> 00:39:59,130 It's the easiest to get your hands on. 861 00:39:59,130 --> 00:40:04,120 s it's the least sensitive because of the low energy 862 00:40:04,120 --> 00:40:05,640 that's released. 863 00:40:05,640 --> 00:40:08,290 OK, so the other thing that I think 864 00:40:08,290 --> 00:40:11,200 is amazing about the phosphorimager is, 865 00:40:11,200 --> 00:40:16,450 if you look at the linearity of detection, 866 00:40:16,450 --> 00:40:20,590 it's linear over five or six orders of magnitude, 867 00:40:20,590 --> 00:40:23,290 whereas, in the old days, you used to use some kind of film 868 00:40:23,290 --> 00:40:24,160 on top. 869 00:40:24,160 --> 00:40:27,850 And the film would absorb the radioactive decay 870 00:40:27,850 --> 00:40:29,680 and make a spot. 871 00:40:29,680 --> 00:40:31,450 And that was linear over a period 872 00:40:31,450 --> 00:40:34,400 of over one order of magnitude. 873 00:40:34,400 --> 00:40:35,650 So you had nonlinearity. 874 00:40:35,650 --> 00:40:38,440 So that was really hard to do quantitation. 875 00:40:38,440 --> 00:40:41,170 So phosphorimager have revolutionized 876 00:40:41,170 --> 00:40:45,400 what you can do in terms of analyzing TLC or gels like Liz 877 00:40:45,400 --> 00:40:47,020 talked about today in class. 878 00:40:47,020 --> 00:40:50,050 OK, so what we need then is a quantitative way 879 00:40:50,050 --> 00:40:54,410 of actually measuring radioactivity. 880 00:40:54,410 --> 00:41:02,140 And what is the standard for radioactivity we use? 881 00:41:02,140 --> 00:41:10,845 And so the quantitation and the standard is called a curie. 882 00:41:14,312 --> 00:41:16,950 It also could be called becquerel 883 00:41:16,950 --> 00:41:20,370 after the discovery of radioactivity. 884 00:41:20,370 --> 00:41:24,630 And there's a relationship between the two. 885 00:41:24,630 --> 00:41:27,900 And what we know, the standard of radioactivity with the Curie 886 00:41:27,900 --> 00:41:32,610 is defined as the substance that decays at 3.7 times 10 887 00:41:32,610 --> 00:41:35,130 to the 10th disintegrations per second. 888 00:41:35,130 --> 00:41:45,000 So one curie equals 3.7 times 10 to the 10th disintegrations 889 00:41:45,000 --> 00:41:47,190 per second. 890 00:41:47,190 --> 00:41:54,540 Or the number that you often see is 2.2 times 10 891 00:41:54,540 --> 00:41:58,210 to the 12th disintegrations per minute. 892 00:41:58,210 --> 00:42:00,420 So this is often what you see on the bottles 893 00:42:00,420 --> 00:42:05,120 when you actually buy radiolabeled material. 894 00:42:05,120 --> 00:42:11,107 OK, so again, what you see is you're counting. 895 00:42:11,107 --> 00:42:12,690 Efficiency, as I've already described, 896 00:42:12,690 --> 00:42:15,697 varies with the energy that's released. 897 00:42:15,697 --> 00:42:17,280 And you have to think about quenching. 898 00:42:17,280 --> 00:42:20,980 That was just repeating what I've already told you. 899 00:42:20,980 --> 00:42:23,550 OK, and so then what do you do? 900 00:42:23,550 --> 00:42:26,280 So when you purchase radioactivity, 901 00:42:26,280 --> 00:42:27,690 how does it come? 902 00:42:27,690 --> 00:42:30,760 OK, so you guys are used to purchasing something from Sigma 903 00:42:30,760 --> 00:42:32,400 or Aldrich or wherever you get it. 904 00:42:32,400 --> 00:42:35,040 You look at it, and you can see something in the bottle. 905 00:42:35,040 --> 00:42:38,490 When you purchase radioactivity, you can't see anything. 906 00:42:38,490 --> 00:42:39,390 Why? 907 00:42:39,390 --> 00:42:42,425 Because there's no material, almost no material 908 00:42:42,425 --> 00:42:43,050 in your bottle. 909 00:42:43,050 --> 00:42:45,700 It's all radioactivity. 910 00:42:45,700 --> 00:42:47,940 So if you put it in a scintillation counter, 911 00:42:47,940 --> 00:42:52,590 you would have, you know, 10 to the ninth decompositions 912 00:42:52,590 --> 00:42:55,050 per minute, OK, but no material. 913 00:42:55,050 --> 00:42:58,230 So you can't work with it because you can't weigh it. 914 00:42:58,230 --> 00:42:59,860 You can't do anything with it. 915 00:42:59,860 --> 00:43:02,790 OK, you have-- I don't know-- a picomole of material. 916 00:43:02,790 --> 00:43:05,790 It depends on the material that you buy. 917 00:43:05,790 --> 00:43:08,880 So the question is what do you do with this material 918 00:43:08,880 --> 00:43:09,820 when you get it. 919 00:43:09,820 --> 00:43:11,320 Well, you want to be able to use it. 920 00:43:11,320 --> 00:43:14,690 And in our case, how are we using it? 921 00:43:14,690 --> 00:43:18,960 We're going to buy GTP that's gamma P-32-labeled. 922 00:43:18,960 --> 00:43:21,910 To be able to use this, we need to measure something. 923 00:43:21,910 --> 00:43:23,645 So what is the first thing we do? 924 00:43:23,645 --> 00:43:24,645 Has anybody got a guess? 925 00:43:28,020 --> 00:43:31,880 You can't use what you buy because what you buy 926 00:43:31,880 --> 00:43:35,970 is you'll get a little vial like this. 927 00:43:35,970 --> 00:43:38,810 And that's what you see, or you might 928 00:43:38,810 --> 00:43:41,620 be able to see some red material that's 929 00:43:41,620 --> 00:43:43,080 decomposed material actually. 930 00:43:43,080 --> 00:43:43,580 Yeah? 931 00:43:43,580 --> 00:43:46,670 AUDIENCE: You need like a kinase that'll 932 00:43:46,670 --> 00:43:50,030 exchange the phosphate with the radioactive phosphate. 933 00:43:50,030 --> 00:43:52,330 JOANNE STUBBE: No, I mean, you could 934 00:43:52,330 --> 00:43:54,920 do that if you wanted to convert it into something else. 935 00:43:54,920 --> 00:43:57,970 But we want the gamma P-32-labeled ATP. 936 00:43:57,970 --> 00:44:00,280 That's what we want to use in our assay. 937 00:44:00,280 --> 00:44:03,286 So what do you do to make this usable? 938 00:44:03,286 --> 00:44:04,542 AUDIENCE: You add some buffer. 939 00:44:04,542 --> 00:44:05,500 JOANNE STUBBE: Do what? 940 00:44:05,500 --> 00:44:06,850 AUDIENCE: You add some buffer to the [INAUDIBLE].. 941 00:44:06,850 --> 00:44:08,308 JOANNE STUBBE: You add some buffer. 942 00:44:08,308 --> 00:44:11,310 OK, does that change the amount of material? 943 00:44:11,310 --> 00:44:13,487 No, so we probably do have some buffer, OK, 944 00:44:13,487 --> 00:44:15,820 because we want to be able to transfer it into something 945 00:44:15,820 --> 00:44:17,440 so we can do our assays. 946 00:44:17,440 --> 00:44:18,368 So go ahead. 947 00:44:18,368 --> 00:44:20,326 AUDIENCE: Yeah, then we're going to transfer it 948 00:44:20,326 --> 00:44:22,451 when you have a specimen that you are going to take 949 00:44:22,451 --> 00:44:23,897 some buffer [INAUDIBLE]. 950 00:44:23,897 --> 00:44:25,480 JOANNE STUBBE: OK, so you can, but you 951 00:44:25,480 --> 00:44:26,830 have no material in there. 952 00:44:26,830 --> 00:44:29,230 So if you had a substrate that was 953 00:44:29,230 --> 00:44:32,050 10 to the minus 12th molar in solution, 954 00:44:32,050 --> 00:44:34,910 would the enzyme ever turn it over? 955 00:44:34,910 --> 00:44:36,790 Probably not, because it could never find it. 956 00:44:36,790 --> 00:44:38,390 OK, so that's not going to work. 957 00:44:38,390 --> 00:44:40,000 So what is the-- go ahead. 958 00:44:40,000 --> 00:44:41,120 What would you do? 959 00:44:41,120 --> 00:44:42,970 AUDIENCE: Like would it matter if like 960 00:44:42,970 --> 00:44:44,820 the radiolabeled phosphorus were just 961 00:44:44,820 --> 00:44:46,537 like a fraction of regular phosphorus? 962 00:44:46,537 --> 00:44:48,400 Like could you add some like unlabeled phosphorus? 963 00:44:48,400 --> 00:44:50,608 JOANNE STUBBE: Exactly, and so this is the key point. 964 00:44:50,608 --> 00:44:53,050 The first thing you do is you take unlabeled material, 965 00:44:53,050 --> 00:44:56,280 and you add it into the radiolabeled material. 966 00:44:56,280 --> 00:44:59,690 And how much you add depends on what you're using it for. 967 00:44:59,690 --> 00:45:01,570 So if you're going to use assays, 968 00:45:01,570 --> 00:45:04,510 and you don't need a very sensitive method, 969 00:45:04,510 --> 00:45:05,786 you can add much more. 970 00:45:05,786 --> 00:45:07,660 If you're going to look at a binding consent, 971 00:45:07,660 --> 00:45:09,910 you know you're pushing a lower limit of detection 972 00:45:09,910 --> 00:45:13,090 because you have some estimate of the number of receptors. 973 00:45:13,090 --> 00:45:15,070 Then you would add much less. 974 00:45:15,070 --> 00:45:17,410 So what you're going to do then-- the first thing 975 00:45:17,410 --> 00:45:20,170 you do when you get radioactivity 976 00:45:20,170 --> 00:45:24,010 is you add unlabeled material. 977 00:45:26,395 --> 00:45:28,770 And I think this is something, if you didn't get anything 978 00:45:28,770 --> 00:45:33,300 out else out of today's discussion, 979 00:45:33,300 --> 00:45:35,340 I think most people won't get this. 980 00:45:35,340 --> 00:45:37,140 When you work with radioactivity, 981 00:45:37,140 --> 00:45:41,440 most of material, one molecule, only one molecule in 10 982 00:45:41,440 --> 00:45:44,520 to the sixth to 10 to the ninth is radioactive. 983 00:45:44,520 --> 00:45:47,080 All the rest are non-radioactive. 984 00:45:47,080 --> 00:45:50,880 OK, so this is just telling you about the sensitivity 985 00:45:50,880 --> 00:45:51,570 of the method. 986 00:45:51,570 --> 00:45:55,240 Somehow using a scintillation counter 987 00:45:55,240 --> 00:45:57,480 or using these phosphorimagers, you 988 00:45:57,480 --> 00:46:02,210 can quantitate the amount of radioactivity you have present. 989 00:46:02,210 --> 00:46:06,750 So when you're dealing with radio label, most of it 990 00:46:06,750 --> 00:46:07,620 is unlabeled. 991 00:46:07,620 --> 00:46:09,925 OK, so what does that tell you then? 992 00:46:09,925 --> 00:46:14,250 So again, the amount of stuff is tiny. 993 00:46:14,250 --> 00:46:19,170 When you add cold material, what does that allow you to do? 994 00:46:19,170 --> 00:46:22,230 What that allows you to do is measure. 995 00:46:22,230 --> 00:46:24,060 And this is the key take-home message. 996 00:46:24,060 --> 00:46:33,940 Now you can measure the specific activity of your material. 997 00:46:33,940 --> 00:46:41,000 OK, so you bought radiolabel. 998 00:46:41,000 --> 00:46:42,260 Let's say tritium. 999 00:46:42,260 --> 00:46:47,555 And then you added protonated material. 1000 00:46:50,440 --> 00:46:52,760 And the specific activity is the amount 1001 00:46:52,760 --> 00:46:56,889 of radioactivity per the amount of material 1002 00:46:56,889 --> 00:46:59,180 that you have present, the number of moles of material. 1003 00:46:59,180 --> 00:47:02,660 So it's in decompositions per minute per micromolar, 1004 00:47:02,660 --> 00:47:05,720 decompositions per nanomole. 1005 00:47:05,720 --> 00:47:09,380 And again, you have to change everything 1006 00:47:09,380 --> 00:47:12,900 to accommodate quenching effects. 1007 00:47:12,900 --> 00:47:15,320 So what you measure from a scintillation counter 1008 00:47:15,320 --> 00:47:18,200 is counts per minute, which is just decomposition 1009 00:47:18,200 --> 00:47:19,370 per minute times quenching. 1010 00:47:19,370 --> 00:47:22,590 So if there's 50%, you see half as much 1011 00:47:22,590 --> 00:47:24,020 as you should be seeing. 1012 00:47:24,020 --> 00:47:27,230 So specific activity is given in counts 1013 00:47:27,230 --> 00:47:31,010 per minute per amount, which is usually 1014 00:47:31,010 --> 00:47:35,270 in micromoles or nanomoles. 1015 00:47:35,270 --> 00:47:40,740 So if you know you have 1,000 counts per minute per nanomole, 1016 00:47:40,740 --> 00:47:46,490 and you count 100 counts, how many nanomoles do you have? 1017 00:47:46,490 --> 00:47:48,920 So you're given your specific activity. 1018 00:47:48,920 --> 00:47:50,390 You do an experiment. 1019 00:47:50,390 --> 00:47:52,940 You have 1,000 counts per minute per nanomole. 1020 00:47:52,940 --> 00:47:55,490 And when you count this-- whoops, when you count this, 1021 00:47:55,490 --> 00:47:58,910 you end up with 100 counts. 1022 00:47:58,910 --> 00:48:00,759 What amount of material do you have? 1023 00:48:00,759 --> 00:48:01,550 AUDIENCE: 21 moles. 1024 00:48:01,550 --> 00:48:02,990 JOANNE STUBBE: Yeah, so that's it. 1025 00:48:02,990 --> 00:48:05,240 So that's the quantitative relationship 1026 00:48:05,240 --> 00:48:10,270 you need to remember to do all these assays that are actually 1027 00:48:10,270 --> 00:48:12,320 in the paper that was described. 1028 00:48:12,320 --> 00:48:15,240 So let me just give you two examples of this. 1029 00:48:15,240 --> 00:48:16,580 We're already late. 1030 00:48:16,580 --> 00:48:18,200 But so this is tritium. 1031 00:48:18,200 --> 00:48:22,520 OK, does anybody see anything weird with tritiated cytidine. 1032 00:48:22,520 --> 00:48:26,090 So this was taken off of Google from Sigma. 1033 00:48:26,090 --> 00:48:28,700 You can buy this from Sigma now. 1034 00:48:28,700 --> 00:48:30,780 Do you think that's reasonable that we 1035 00:48:30,780 --> 00:48:33,710 CT3 in our methyl group? 1036 00:48:33,710 --> 00:48:34,760 So T is for tritium. 1037 00:48:39,520 --> 00:48:42,470 What did I just tell you about our material? 1038 00:48:46,190 --> 00:48:48,140 How much material has got a label in it? 1039 00:48:48,140 --> 00:48:48,740 How much-- 1040 00:48:48,740 --> 00:48:50,285 AUDIENCE: One in 10 the the fourth. 1041 00:48:50,285 --> 00:48:52,910 JOANNE STUBBE: Yeah, so we don't have very much that's labeled. 1042 00:48:52,910 --> 00:48:55,100 Say we had 100% labeled. 1043 00:48:55,100 --> 00:48:57,280 Do you think that would be an issue? 1044 00:48:57,280 --> 00:49:00,670 Say we had a million to-- 1045 00:49:00,670 --> 00:49:03,350 10 to the sixth to 10 to the ninth more tritium. 1046 00:49:03,350 --> 00:49:06,450 What do you think that might do in terms of energy? 1047 00:49:06,450 --> 00:49:06,950 Yeah? 1048 00:49:06,950 --> 00:49:08,375 AUDIENCE: You said tritium is much weaker. 1049 00:49:08,375 --> 00:49:10,180 We're talking about phosphorus here. 1050 00:49:10,180 --> 00:49:12,340 So that's like a huge signal, whereas-- 1051 00:49:12,340 --> 00:49:13,840 JOANNE STUBBE: So even with tritium, 1052 00:49:13,840 --> 00:49:15,500 OK, you still get enough energy. 1053 00:49:15,500 --> 00:49:19,130 If you tried to put that much tritium in your molecule, 1054 00:49:19,130 --> 00:49:21,900 within as fast as you could isolate the material, 1055 00:49:21,900 --> 00:49:24,140 it would be completely decomposed. 1056 00:49:24,140 --> 00:49:27,110 So there are ways to put tritium into the molecule, 1057 00:49:27,110 --> 00:49:30,500 but the decay would completely destroy your molecule 1058 00:49:30,500 --> 00:49:32,310 because you have so much radioactivity. 1059 00:49:32,310 --> 00:49:35,840 So this, which is on the web, is completely incorrect. 1060 00:49:35,840 --> 00:49:39,350 So what you have is one molecule in 10 to the sixth 1061 00:49:39,350 --> 00:49:42,260 that actually has tritium labeled. 1062 00:49:42,260 --> 00:49:44,360 And how much you have, you don't know. 1063 00:49:44,360 --> 00:49:47,230 What you need to do is add cold material, 1064 00:49:47,230 --> 00:49:50,030 and then you need to figure out a way 1065 00:49:50,030 --> 00:49:53,780 to quantitate the amount of material, leucine or GTP. 1066 00:49:53,780 --> 00:49:56,350 And then you count that amount of material. 1067 00:49:56,350 --> 00:49:59,730 And that gives you the specific activity. 1068 00:49:59,730 --> 00:50:01,200 So let me just say one more thing. 1069 00:50:01,200 --> 00:50:03,200 Those of you who have to go, I'm sorry I'm late. 1070 00:50:03,200 --> 00:50:07,640 You can go So where do you get radiolabeled material from? 1071 00:50:07,640 --> 00:50:10,070 Do you think this is easy? 1072 00:50:10,070 --> 00:50:11,570 I mean you could buy leucine. 1073 00:50:11,570 --> 00:50:13,569 I just showed you we could find that on the web. 1074 00:50:13,569 --> 00:50:17,190 You can buy a gamma P-32-labeled GTP as well. 1075 00:50:17,190 --> 00:50:19,174 Most things you can't buy. 1076 00:50:19,174 --> 00:50:22,190 OK, so this is what distinguishes a chemist 1077 00:50:22,190 --> 00:50:26,480 from a biologist in many cases because I could make things 1078 00:50:26,480 --> 00:50:29,300 radiolabeled decades ago. 1079 00:50:29,300 --> 00:50:32,324 Doing a 15-step synthesis, I was able to make molecules 1080 00:50:32,324 --> 00:50:33,740 that allowed me to study something 1081 00:50:33,740 --> 00:50:36,020 that nobody else could study. 1082 00:50:36,020 --> 00:50:38,600 So the question is you need to make your label 1083 00:50:38,600 --> 00:50:41,220 and put it in a specific position. 1084 00:50:41,220 --> 00:50:42,590 And so what do you start with? 1085 00:50:42,590 --> 00:50:45,980 You start with something that's easy to work with. 1086 00:50:45,980 --> 00:50:48,260 And you try to put the label in at the very end 1087 00:50:48,260 --> 00:50:49,190 of your synthesis. 1088 00:50:49,190 --> 00:50:52,190 And one of the things that you often start with 1089 00:50:52,190 --> 00:50:54,350 is sodium borotritiride. 1090 00:50:54,350 --> 00:50:56,570 Why would you start with sodium borotritiride? 1091 00:50:56,570 --> 00:51:00,650 What can sodium borohydride do? 1092 00:51:00,650 --> 00:51:01,670 This is frequently used. 1093 00:51:01,670 --> 00:51:03,590 We'll see this used later on. 1094 00:51:03,590 --> 00:51:05,851 Anybody remember what sodium borohydride does? 1095 00:51:05,851 --> 00:51:06,350 Yeah? 1096 00:51:06,350 --> 00:51:07,340 AUDIENCE: It's reductive. 1097 00:51:07,340 --> 00:51:08,923 JOANNE STUBBE: Yeah, it's a reductant. 1098 00:51:08,923 --> 00:51:11,630 So you can reduce a ketone or an aldehyde to an alcohol. 1099 00:51:11,630 --> 00:51:15,410 OK, so that's frequently used to put in tritium. 1100 00:51:15,410 --> 00:51:18,830 So what are the issues with sodium borohydride? 1101 00:51:18,830 --> 00:51:20,630 Again, this is something that you need 1102 00:51:20,630 --> 00:51:22,100 to think about the chemistry. 1103 00:51:22,100 --> 00:51:26,060 What are the issues with sodium borohydride? 1104 00:51:26,060 --> 00:51:29,600 If you're going to put your label in, OK, I just told you. 1105 00:51:29,600 --> 00:51:32,360 How much of your sodium borohydride is labeled? 1106 00:51:36,656 --> 00:51:38,030 What do you have mostly in there? 1107 00:51:38,030 --> 00:51:43,076 Do you have NaBT4? 1108 00:51:43,076 --> 00:51:48,710 No, so what do we know about tritium versus hydrogen? 1109 00:51:48,710 --> 00:51:51,440 I guess this might depend on how much organic chemistry you've 1110 00:51:51,440 --> 00:51:54,160 had. 1111 00:51:54,160 --> 00:51:56,490 Tritium versus hydrogen, what's the difference? 1112 00:51:56,490 --> 00:52:01,000 Two neutrons, OK, but it's huge in terms of weight, 1113 00:52:01,000 --> 00:52:04,740 OK, because neutrons are the same weight as the protons. 1114 00:52:04,740 --> 00:52:07,840 So what you see is an isotope effect on the reaction. 1115 00:52:07,840 --> 00:52:09,830 So when you use sodium borotritiride, 1116 00:52:09,830 --> 00:52:12,620 the activity is never the same as what 1117 00:52:12,620 --> 00:52:13,640 you got out of a bottle. 1118 00:52:13,640 --> 00:52:15,029 You have an isotope effect. 1119 00:52:15,029 --> 00:52:17,570 The other thing is, if any of you have ever worked with this, 1120 00:52:17,570 --> 00:52:19,319 and you're doing this in aqueous solution, 1121 00:52:19,319 --> 00:52:21,800 what does sodium borotritiride do? 1122 00:52:21,800 --> 00:52:22,940 Anybody got any ideas? 1123 00:52:27,410 --> 00:52:31,458 In water, at pH 7. 1124 00:52:31,458 --> 00:52:32,900 AUDIENCE: Proton exchange. 1125 00:52:32,900 --> 00:52:35,504 JOANNE STUBBE: Proton exchange. 1126 00:52:35,504 --> 00:52:36,940 AUDIENCE: You get hydrogen gas. 1127 00:52:36,940 --> 00:52:37,990 JOANNE STUBBE: You get hydrogen gas. 1128 00:52:37,990 --> 00:52:38,865 You get hydrogen gas. 1129 00:52:38,865 --> 00:52:41,530 The whole little flask would hit you in the face 1130 00:52:41,530 --> 00:52:43,870 with the hydrogen coming off when you're in-- 1131 00:52:43,870 --> 00:52:45,010 and what would you get? 1132 00:52:45,010 --> 00:52:48,020 You'd get a face full of tritium, tritiated hydrogen. 1133 00:52:48,020 --> 00:52:50,440 OK, tritiated hydrogen is not so bad 1134 00:52:50,440 --> 00:52:52,570 because it's not very soluble. 1135 00:52:52,570 --> 00:52:54,970 So it goes into your system and gets washed out. 1136 00:52:54,970 --> 00:52:57,640 If you were producing tritiated water, that's bad. 1137 00:52:57,640 --> 00:52:59,590 So that's the other place where you do this. 1138 00:52:59,590 --> 00:53:02,620 You can get very hot labeled tritiated water. 1139 00:53:02,620 --> 00:53:04,720 And that you have to be really careful of because, 1140 00:53:04,720 --> 00:53:07,470 if you breathe that in, it gets mixed with all 1141 00:53:07,470 --> 00:53:08,680 the unlabeled materials. 1142 00:53:08,680 --> 00:53:11,720 And it takes forever to get rid of it. 1143 00:53:11,720 --> 00:53:14,860 So I think we're not even going to get to-- 1144 00:53:17,425 --> 00:53:19,240 I'd let you go through all of this. 1145 00:53:19,240 --> 00:53:21,970 But what I want you to do now is go back and think 1146 00:53:21,970 --> 00:53:24,070 about what this data means. 1147 00:53:24,070 --> 00:53:26,740 At least you now know what the assays are. 1148 00:53:26,740 --> 00:53:30,180 And think about the axes. 1149 00:53:30,180 --> 00:53:34,200 And think about, you know, cognate versus near-cognate. 1150 00:53:34,200 --> 00:53:36,240 Why do we see a lag here? 1151 00:53:36,240 --> 00:53:37,860 What happens at 100%? 1152 00:53:37,860 --> 00:53:39,360 You're using up all the GTP. 1153 00:53:39,360 --> 00:53:40,207 What does that mean? 1154 00:53:40,207 --> 00:53:41,790 That's what I want you to think about. 1155 00:53:41,790 --> 00:53:44,490 If you come over here, and you're looking at dipeptide, 1156 00:53:44,490 --> 00:53:45,510 not tRNA. 1157 00:53:45,510 --> 00:53:47,940 We're looking at a dipeptide. 1158 00:53:47,940 --> 00:53:50,312 You need to look at the axes. 1159 00:53:50,312 --> 00:53:51,520 They're completely different. 1160 00:53:51,520 --> 00:53:52,500 One is micromole. 1161 00:53:52,500 --> 00:53:53,820 One is nanomole. 1162 00:53:53,820 --> 00:53:56,340 So we're trying to get you to actually look 1163 00:53:56,340 --> 00:53:58,320 at the primary data, which you may or may not-- 1164 00:53:58,320 --> 00:54:01,510 how many saw this difference when you read the paper? 1165 00:54:01,510 --> 00:54:03,570 OK, so to me, this is what we're trying to get 1166 00:54:03,570 --> 00:54:05,430 you to do on the first test. 1167 00:54:05,430 --> 00:54:08,190 I can tell you a lot of people have trouble looking at this. 1168 00:54:08,190 --> 00:54:09,940 That's what we're trying to get you to do. 1169 00:54:09,940 --> 00:54:12,470 That's why we're going through this in so much detail. 1170 00:54:12,470 --> 00:54:14,157 And then it becomes second nature. 1171 00:54:14,157 --> 00:54:15,240 You just start reading it. 1172 00:54:15,240 --> 00:54:16,290 You look at the details. 1173 00:54:16,290 --> 00:54:17,625 And you make a judgment. 1174 00:54:17,625 --> 00:54:20,250 If you don't understand what's going on, you go look it up, 1175 00:54:20,250 --> 00:54:22,320 or you go talk to somebody about what 1176 00:54:22,320 --> 00:54:25,370 the issues are with the method. 1177 00:54:25,370 --> 00:54:29,530 Here again, the lag phases are not all that different. 1178 00:54:29,530 --> 00:54:33,060 But here, if you take the differences in amounts 1179 00:54:33,060 --> 00:54:37,140 into account, you're only getting 1%, 1% to 2% the amount 1180 00:54:37,140 --> 00:54:40,170 of leucine incorporated into the peptide 1181 00:54:40,170 --> 00:54:41,940 as you would with two phenylalanines. 1182 00:54:41,940 --> 00:54:43,650 And that's because it's a near-cognate. 1183 00:54:43,650 --> 00:54:45,090 And what's happening? 1184 00:54:45,090 --> 00:54:48,300 You know, you're having discrimination 1185 00:54:48,300 --> 00:54:51,730 between peptide bond formation and dissociation. 1186 00:54:51,730 --> 00:54:55,150 So that's the proofreading part of the overall mechanism. 1187 00:54:55,150 --> 00:54:58,740 So I think thinking about these two slides 1188 00:54:58,740 --> 00:55:01,560 really tells you quite a bit about whether you believe 1189 00:55:01,560 --> 00:55:04,680 the model that Rodnina-- 1190 00:55:04,680 --> 00:55:08,220 whether the model is reasonable given the data 1191 00:55:08,220 --> 00:55:08,935 you actually see. 1192 00:55:08,935 --> 00:55:09,810 All right, I'm sorry. 1193 00:55:09,810 --> 00:55:10,430 I'm way over. 1194 00:55:10,430 --> 00:55:13,430 So I'm going to stop here.