Oliver Smithies:[00:00:00] This is a start on Book I, 1962, and a quick glance at the book looks as if it’s all directed towards understanding how to resynthesize, or a great deal of it, how to resynthesize the polymers after dissociation with mercaptoethanol. It begins with acetone and mercaptoethanol resynthesis, page 1, Thursday, June the 14th, with the result on page 3, essentially identical acetone result with the previous. Mercaptoethanol, no hemoglobin binding protein smear.
Several repeats on page 5 [00:01:00] with a comment that, “This result again, compare Book H, page 150, shows acetone leaving, or giving haptoglobin 2-2 polymers, which are not hemoglobin binding — non hemoglobin benzidine positive. Abandon acetone for the time being.”
Heat dissociation with [Sustain?] on Monday, June 18th, page 9, trying to find the magic formula. Try annealing, like DNA, [00:02:00] page 11. (laughter) Usual comment, “Looks very promising indeed.” I was a perpetual optimist.
Heat-tests, June 20th. So is it haptoglobin 1-1 is OK at 60 degrees, but 2-2 is about 80-90% wrecked at higher temperatures. At 70 degrees, 1-1 is still more than 85% preserved; 2-2 is about 95% wrecked by heating in the presence of [00:03:00] just a buffer with Tris and EDTA. And notice EDTA now, not Versene. And with a comment on page 15, that “Results of heat tests show 2-2 is stable, SH at 50 degrees, that is partly-broken down at 50, plus SH.” That’s stable minus SH at 50, but partly broken down at 50 degrees plus SH. [00:04:00]
Slow dialysis from urea tests on June 25th, Monday, page 23. For once, “The results are disappointing. Better resynthesis on first dialysis, return to anaerobic resynthesis after urea acetone, high pH.” Still haven’t given up on acetone despite the comment.
High mercaptoethanol tests, June 26th. [00:05:00] Anaerobic tests with acetone at a higher pH. Whole set of gels on them, on page 31.
Trying to make it anaerobic on page 33. [00:06:00] And there’s no doubt from these gels that there is good splitting, although there isn’t any particular comment about it. Though the idea of anaerobic urea resynthesis by Sephadex removal of the urea, G-25 Sephadex is being pursued on page 37.
On page 39, the comment then being that [00:07:00] page I-37, “Anaerobics are poorer than the corresponding aerobic samples, but 1-1 plus 2-2 is not giving good 2-1. Definitely mercaptoethanol for the latter part of the resynthesis is not desireable.” Miscellaneous anaerobic repeats on page 41.
Oxidation tests were carried out on Thursday, July 5th, [00:08:00] with samples that were straight from under the oil, and all smell of mercaptoethanol, and then with air oxidation, all doubtful of any smells, but the results show even this brief oxidation period has an effect. There’s more hemoglobin binding in the 1-1 plus 2-2, and the 2-2 plus mercaptoethanol samples, after air. So beginning to try to get things to go back by oxidation again. Or not beginning, continuing. [00:09:00]
Anaerobic tests with acetone. No, anaerobic tests, but mercaptoethanol alone on page 45. And see, nothing very promising. We’re coming to the comment on page 44 that, “No effective oxidation. Must use something other than mercaptoethanol for the final resynthesis.” So, still fiddling with acetone on page 147.
With a general review on page 49, Monday July 9th, that it’s clear that 0.02M mercaptoethanol does 2-2 harm, although further oxidation [00:10:00] may clear up the trouble,” et cetera. Sodium sulfide, again.
Tuesday July 10th, page 51, “Feel to be making some progress here, because the results show that 0.001M sodium sulfide is very good, and 0.001M mercaptoethanol [00:11:00] almost as good.” But they’re getting rid of the polymers. This is just getting rid of the polymers again, not their resynthesis.
Something interesting on page 53, is a continuation of the comments on the bottom of page 51. It’s saying that the control with 1-1 plus 2-2 for a urea test, and also with 0.01M sulfite, in view of the reactions. And here are the beginning of the reactions which later on proved to be very important in understanding what was going on. So I have a diagram [00:12:00] here of a polypeptide joined with a disulfide bond, which in the presence of mercaptoethanol would give SSME and SH, which with stronger mercaptoethanol, would give SH on both Sustains. So the mixed disulfide present on the top right-hand diagram of 53 shows that I am at least beginning to think about mixed disulfides, and also the possibility later on of the sulfite reaction at the bottom of the page where it’s SS treated with sulfite at any concentration [00:13:00] with urea will go to S-SO3- and S- at any concentration in the presence of urea, and might be reversed. This is a [Rupert-Cecil?] reaction which I was aware of, and S-SO3- product. But here is the beginning of the chemistry which later was successful in solving it, but I don’t know how well I’ve recognized it at this point. At least I’ve written it down.
Various tests on page 57. Results show some slight improvement, but essentially [00:14:00] no change. Beginning to think about repeating the 2-2 SO3 sulfite experiment, since it’s clear that splitting occurs OK. Maybe we can reverse things. Some discussion of this on page 63, still, with sulfite and with acetone, and with sodium sulfide, with the comment that, “The results suggest that 25% acetone didn’t split [00:15:00], that the results with sodium sulfide and with SO3– are identical,” et cetera. “Beginning to think that both urea and acetone act by removing SH from the reaction mixture. Back for other reagents that cannot react.” Remembering then that reaction earlier, getting the thioether with acetone and mercaptan.
Various repeats on page 65. Results show no essential difference, again, for aerobic versus anaerobic. No for sulfite versus SH-, although SO3– may be [00:16:00] a trace better. Struggling, struggling. Many of these gels look identical to what have been on previous pages.
pH tests for resynthesis on page 73, no hemoglobin binding. Results of I-55, [00:17:00] very poor electrophoresis due to bridge siphoning. But the 1-1 plus 2-2 plus SO3– is quite good, in terms of what is happening, though looking at it at this date — and I think maybe that’s the way I also felt, because on the following page, Thursday July 20th, on page 77, I say, “In view of very poor improvement of all sample in the presence of zero or very low [00:18:00] thiol concentrations, I decided to attempt improvement of several past experiments by using 0.01M mercaptoethanol aerobically,” et cetera, and some anaerobic things.
The sulfite reaction seems to be cheering me up on page 79. “Page I-55, SO3– is markedly improved with mercaptoethanol.” That was I-55 SO3 1-1 plus [00:19:00] 2-2, but I-55 2-2 SO3 is not so good.
Repeat of pH tests on page 83, not very informative.
I didn’t ever seem to give up. [00:20:00] Page 91, retest once more. Magnesium, and Mg+, plus ferric and copper, and on page 95, tests of metals. None of them had any effect that’s visible. These books need a lot of attention. [00:21:00]
Mercaptoethanol plus or minus and dialysis, page 99. Dithiodiglycol dialysis tests. I know now that this is the beginning of, again, the use of dithiodiglycol, but I’m not sure that the importance of it is going to be recognized right away.
Saturday, July 28th. This was dithiodiglycol dialyzed, and B-40 dialyzed. There wasn’t any obvious difference [00:22:00] looking at it. Benzidine dithiodiglycol is better than B-40, better than mercaptoethanol. Looking at it at this later date, it looks as if nothing has really happened.
Free radical tests on Sunday, July 29th, page 109. Dithiodiglycol, and added hydroquinone [00:23:00] 1000M. Results show that dithioglycol and hydroquinone are poor. And with a comment that, “Almost certainly, I’ve been misled, incorrectly mislead, in recent tests by thiol interfering with a benzidine reaction. Return to complete test,” et cetera,” but first check GSSG for removal –“ that’s the dithiodiglycol, “– for [00:24:00] removal of spurious zone.”
Still slowly getting to useful thoughts on what might be going on, on page 113, August 3rd, general review of SH groups in alpha chains, showing that 1F-alpha plus mercaptoethanol, three SH groups, with sodium sulfide, one disulfide bond that’s left, and one SH is free, and with mercaptoethanol, all of them are — it’s varying degrees of cleavage. It’s a little difficult to understand, looking at it again, [00:25:00] I’ll try again. Page 113, it’s a general review of the thiol groups in the alpha chains. Analyses show that 1F-alpha and 1S-alpha have three SH groups, three sustains, and two alpha, as six, but usually one pair in the six is already a disulfide, and this is a concentrated in J, the junction peptide. And so this suggests the following arrangement, that 1F alpha plus mercaptoethanol gives a linear chain with three SH groups free, sustains. 1F alpha plus sodium sulfide cleaves — gives a one SH [00:26:00] group, and a disulfide bond still left. Where, in two alpha plus mercaptoethanol has four sustains reduced, and one disulfide bond still left, et cetera, and two alpha per sodium sulfide has two disulfides left, and two SHs, so various combinations of which ones are cleaved and which are not. None of the previous book Roman-21 tests contradict this, also tests plus or minus iodoacetic acid suggest that HP, that 1F-alpha and 1S-alpha lose two charges with iodoacetic, whereas 2-alpha loses four charges. [00:27:00] So therefore, it’s essential to open out the 2-alpha fully if possible.
And going on with this thought on the following page, with a prediction of what I might hope to see with an 8M urea, 0.05, 0.01, 120% formic acid gel.
And the gel on page 117, actually looks quite promising, looking at it [00:28:00] from here. It doesn’t actually say whether I thought it was good or not. Ah, yes. It’s difficult to read, that’s all, page 117. “The predictions appear to be confirmed, and so now check what happens with this, and not the other.” It is, looking at it, hope to see a diagram, and the result on page 116, are remarkably good. So, I’m surprised that it wasn’t emphasized more that it really was a very good description of what would happen. [00:29:00] Just a comment, “Predictions appear to be confirmed,” is really quite low key for my usual comments.
Some repeats on page 121. Results reviewed on page 120. The book order seems to be correct. Must have doubted my labeling. [00:30:00] Thinking about column chromatography, [porath?] columns, [with arga?], on page 123 and 125, and 127.
And that goes on to page 131, I’m still talking about what might be expected, corrected. [00:31:00] On page 131, I’m setting up an experiment which gives spectacular looking results on page 133, Thursday September 20th. “Disulfide bond tests in HP alpha chains, 1F, S-alpha, and S, and HP2-alpha,” et cetera, “treated with various things.” And, what I expected to see is on page 132, and you’ll see what the comment is. No specific comment, [00:32:00] surprisingly enough. And, what to expect to see in the gel with mercaptoethanol, and what expected to see in the gel without mercaptoethanol, and two gels are there shown with mercaptoethanol without, both acidic gel [00:33:00], but looking at them from this vantage point, they don’t appear to be different, although there is no comment on the result.
Page 135, testing HP-alpha chain digest with alpha-chymotrypsin. [00:34:00]
On page 137, hemoglobin without tests, neither the acid method nor the alkaline method dissolved this long-set agar, [00:35:00] and only used the supernatant in an attempt to get material from hemoglobin agar, mixed as — trying to make a hemoglobin chain with agar again, going backwards quite a long way, not clear why I decided to try that. [00:36:00] So trying to get hemoglobin-haptoglobin, et cetera, with agar hemoglobin. Then the last few pages of this book are really rather difficult to interpret; I might try again, but they’re obviously related to [00:37:00] binding hemoglobin to agar, and testing various aspects of this. It certainly doesn’t look very promising, and the end of the book occurs on Tuesday, October 9th.