Oliver Smithies:[00:00:00] This is Book N, begins in 1963, April, and continues th‑‑ in that year. It’s going back to immunoglobulins, at the beginning, separation of heavy and light chains, and some ideas for sequencing. (pause) For example, on April — Monday, April 27, page 11, tests of various Bence Jones proteins. And the four proteins that are being considered are [00:01:00] [name redacted], [name redacted], and one called SHBJ and BOBJ and AGBJ, Bence Jones proteins, which, they’re… Looking at the gels, the formic — 8‑molar formic acid gels, with urea and 0.2-molar mercaptoetha– Beautiful separations. And all five Bence Jones proteins have different mobilities in this gel, [name redacted], [name redacted], and the other two — the other three — a very pretty gel, on page 10. Bo‑‑ preparations of the [name redacted] Bence Jones protein, again, starting with nine liters of urine. [00:02:00] Looking at the [merca?]‑‑ the sizes of the Bence Jones proteins, on page 15, by the usual method of varying starch concentrations — with a comment on that page with the… This is the multiple-gel method of trying determine molecular size. “This SHBJ‑2 may be bigger than [name redacted] but the BOBJ‑2 and [name redacted] do not differ significantly.” And BOBJ‑2 was repeated, and May 14th through 16th. So we’ll come back to it at another time. [00:03:00] [name redacted], a new method of preparation. Phillips Bence Jones, a new w‑‑ sample. Looks very thin.
Looking at May 1st, Wednesday, molecular weight determinations on AGBJ‑2 and [name redacted] — recalculated — and repeated on May 8th and 9th. [00:04:00] Trying to determine molecular si– Repeat again, on page 25, [name redacted] versus SHBJ‑2. (pause) Digesting with carboxypeptidase, with DFP-treated carboxypeptidase from Worthington, [00:05:00] using lower concentrations, on page 27, different digesting conditions. More work on these immunoglobulins. Testing of inhibition by gamma globulins split with mercaptoethanol, plus or minus iodoacetamide and sulfite, and with copper, etc., Ma‑‑ Thursday, May 16th — (laughs) with a comment at the bottom, “Stick to mercaptoethanol plus or minus iodoacetamide, at present.”[00:06:00] BJ antibody preparation. “Joel [Solomon?] used 12 rabbits, eventually 9, and injected [name redacted],” purified it, Bence Jones protein. And some results of the immunization — very faint bands. Trying to photograph the results of these tests, on pages [00:07:00] 40 through 43. Trying to do immuno-electrophoresis with these serums, these antiserums, and these Bence Jones proteins, on page 47. General conclusion that — is that “Anti-[name redacted] has a nonspecific antibody against the serum impurity, in addition to anti-[name redacted] Bence Jones. This cross-reacts with [name redacted] serum but not with [name redacted] Bence Jones.” So rather complicated results. I never was very fond of these tests, I remember. [00:08:00] Here is, finally, the image that I’ve been trying to find for some time, of “Haptoglobin finals, repeat,” Friday — Tuesday, May 28, page 51, getting back to more secure territory. It’s a beautiful gels — and photographed on that page. And the image occurs in the — in the paper, I think. Let’s just see if we can find it. (break in audio) Yeah. I know, because… See that… So page 50 in Book N is a very pleasing page, a beautiful large enlargement of the gel, showing the differences between the alpha chains of Hp 1F, 1F, 1F, [00:09:00] 1S, 1S, 1S, 2‑1F, 2‑1S, and 2‑2, which is reproduced as Figure — Plate 1 in the paper that w‑‑ that I wrote with George Connell and Gordon Dixon, on “Gene Action in the Human Haptoglobin‑‑” Journal — Molecular Biology, 1966 — Plate 1. It’s very enjoyable to see. It’s an absolutely glorious gel. And the reproduction in the journal is very good, very pretty. Going on with the book, antibody tests with the antisera, on page 53 and [00:10:00] so on. Getting sa‑‑ another sample of another Bence Jones, [name redacted], prepared on page 59. And haptoglobin repeats, on Saturday, June 1st, but really not needed, after that beautiful one on the earlier page — just confirming the result. Why I had some difficulty recognizing the figure on page 60 versus the figure on page 50. They’re very, very similar. Just 50 is just a slightly better gel. [00:11:00] I know why I knew the difference, because that band is not there, that little trace band, not present here but it… So I thought it was this but… Yeah. So it’s j‑‑ the Saturday, June 1st, page 60 and 61, is a repeat of the beautiful gel on page 50.
Purifying [name redacted] Bence Jones protein, following pages. And we’re checking that there is a difference between [name redacted] and [name redacted] — or [name redacted] and [name redacted], page 64 and 65. [00:12:00] Various preparations of [name redacted] compared. And all are alike. And two preparations of — or, no, one preparation of [name redacted], which is different.
Beginning to think about Edman degradations, now, to do sequencing from the amino terminus, Monday, June the 10th, page 67, this being in 1963. So we had a Rotary Evapo‑Mix, [00:13:00] etc., for coupling. And details of how to do the Edman deg‑‑ or how we were going to do or did the Edman degradations, on Monday, June 10. And there is a chromatography sheet set up.
Page 71, June 11th, Tuesday, tryptic fingerprints of N, C, and J. That’s of the — available from a pilot experiment May 8th, 1963. I need to look [00:14:00] back. (pause) Page 71, again, Tuesday, June 11th, is talking about fingerprints on purified N, C, and J, N‑terminal, C‑terminal, and junction peptide from a separation carried out May 8th. But there is no entry in the book of what was going on at — on May 8th. So it’s no‑‑ rather poorly documented in the [00:15:00] lab noteboo‑‑ in my lab notebook. But I was trying to do fingerprints on the N‑terminal, C‑terminal, and junction peptides again, with a different method of digestion, with… The N, C, and J were chymotryptic, as I remember. And this was now digesting with trypsin, to see if I could get another piece of information.
It is — I think it was about this time that I went to Toronto to learn to do peptides and peptide analysis [00:16:00] on purified material. But we may to come to that in the book. But my history of flying is related to that. Because I went to Gordon Dixon’s lab — I traveled from Wisconsin to Gordon Dixon’s lab, to learn to do the work, to do the same type of work that he’d been doing with peptide analysis and labeling N‑terminal and so on. And when I got there, I found that I just didn’t like doing the work at all. And I decided that I wasn’t going to spend the rest of my life (laughs) doing experiments that I didn’t like. So I went down to Toronto Island airport and took flying lessons. And those are some of the first flying lessons I ever had, at Toronto airport — rather spectacular. I remember that the [00:17:00] instructor, who must have been a young buck… Because he — in demonstrating for an early student, he s‑‑ he — we did a spin, which is something that is usually reserved for later students, not beginners. Because it’s rather spectacular from —
— inside the cockpit — as I also found out when I was teaching flying. In the glid‑‑ one of my students was so frightened of a — of spinning that he wouldn’t even let me demonstrate it. Or he tried to prevent me from demonstrating it. But I did demonstrate it. But he was very frightened. He’d been badly trained before me. But anyway, that was — that’s the story of going down to Toronto Island airport. I think it’s shortly — in — after this time. I’ll look at my airplane logbook, to see whether the dates coincide with this period. I think they probably do. [00:18:00] But here I was trying to do it in Wisconsin.
So, for example, the ideas on page 83 are, “Consider taking half a gram of Bence Jones protein and carrying through several steps of Edman degradation and then dissolving in urea — in” every pur‑‑ “and in repurifying. Carry small sample one step further,” etc., to see if I could get longer sequences by the Edman degradation. Rather interesting to see an attempt at getting protein sequencing to work, with the Edman method, in my hands. So fingerprints on page 85, and [00:19:00] 87.
Back to a rather happier topic, in many ways, on page 89, Monday, July 22nd, haptoglobin 2‑1 synthesis. Somebody marketed — reported that isozyme can be dissociated and reassociated by freezing and thawing, in 1‑molar sodium chloride solutions. And so I just suggested that a high ionic strength at zero Centigrade during the thawing would concentrate the protein. And sort of tested with — so I decided to test with the haptoglobins, using 1% 1‑1 and 2‑2, and the usual mixture of 1‑1 and 2‑2, [00:20:00] all in 1‑molar sodium chloride — and then dilute one, and testing. The — and then — but the results are very clear that nothing happened. The freezing, thawing with mercaptoethanol and the freezing and thawing with me‑‑ without mercaptoethanol, in the presence of 1‑molar sodium chloride had absolutely no effect. (laughs) With a little comment, “No changes in 1‑1 + 2‑2. Repeat with solid sodium chloride.” There is a second gel, which is just a poorer gel. But it doesn’t give any different result.[00:21:00] New fingerprints, on July 23rd — different column, cellulose ethylamine, on page 93, [sodacine?], cellulose, on page 95. Com‑‑ just some checks on various samples, to — on Wednesday, August 7th, u‑‑ regular formic acid, 8‑molar urea gel, with mercaptoethanol. [00:22:00] And one a little more dilute. It is a straightforward type of pattern, no different from previous result. And so the comment is, “Confirms that ‘2,’” in quotation marks, “1S, and 2FS is 2FS and 2SS.” We were beginning to realize that SS could be — that 2 could be — have more than one form. It didn’t have to be FF, FS. Two could FS, FF, or SS. This was with Walter Nance. And later on he wrote a paper on this topic. Which, no doubt we shall come to tests… [00:23:00] And this is June 25th. Just — again, just to go over that again, this related to the work that Walter Nance was doing at that time, where the haptoglobin 2, we realized, would — shouldn’t always be FS but might FF or SF or SS, etc. And so the test is whether the 2 is the same. I’ll have to look up and see what date his work was published. But it wasn’t that time and was a little later. [00:24:00] (pause) Back to haptoglobin resynthesis, Tuesday, October 15th, page 101 — with — talking about a paper by J.L. Peel, where he was catalyzing the oxidation of 0.05-molar mercaptoethanol at 37°. So, Wednesday, October 16, has the title, “Haptoglobin [00:25:00] resynthesis for removal of spurious zones. 0.03-molar mercaptoethanol, pH 7, 37°, plus or minus Factor B,” which is what had been claimed to be catalytic, a v‑‑ it’s a vitamin B12 derivative — to be catalytic for oxidation. So that this — with — was in the presence of Factor B. And the following page, 105, so the samples look remarkably similar in the gel, and are mercaptoethanol, mercaptoethanol plus Factor [00:26:00] B, longer-term,” etc., etc. The results suggest that 1‑1 is no good and that the general results are clear that 0.03-molar mercaptoethanol at 37°, pH 7 will dissociate Hp 2‑2 essentially completely, in 60 minutes. Some resynthesis with Factor B. But, “Must investigate the cleavage more thoroughly. Test anaerobically.” Not anything very convincing, in the gel or in the statement of interpretation. So, page 107, test of haptoglobin 2‑2, mercaptoetha‑‑ lone splitting, semi-anaerobic — [00:27:00] and with a gradient gel of different concentrations of mercaptoethanol, simple effect of mercaptoethanol.
And if we pause a moment, I think that, in fact, is a gel that was used in a later paper. (break in audio) The gel on page 106 is very similar to a gel that I published in — when I finally understood what was happening in the cleavage stages. If I look at Figure 1 in my paper “Disulfide-Bond Cleavage and Formation in Protein,” which is published in Science, December 1965, the gel is [00:28:00] remarkably similar. I’m not sure if it is exactly the correct one. We’ll come back to this topic when I find that gel. No, this gel on page 106 is not the published gel. But it’s a prelude to the published gel. It’s very, very similar — just showing the effect of increasing amounts of mercaptoethanol, in the absence of anything — no urea and no alkylation, just changing hapto‑‑ changing mercaptoethanol concentration, where the polymers dissociate and you can go — you can see that they’re dissociated into something that appears to be a monomer. [00:29:00] But this is with added hemoglobin. The gel on page 106 is free hemoglobin, in the top. And these are hemoglobin-binding. And it appears that, at the lower concentrations of — the intermediate concentrations of mercaptoethanol, there is dissociation into the monomer but, as it — as the concentration increases, the ability to bind with hemoglobin disappears. So this is — it says there that the results are odd. The… “A pre-hemoglobin binding component appears before all the polymers are broken. No stable intermediate, as in yesterday’s experiment. So set up again.” Not completely [00:30:00] understanding. But looking at it from a knowledge of what really is happening, the gel is perfectly rational. But the hemoglobin binding disappears at higher mercaptoethanol concentration. The dissociation from polymers to monomers is — leaves a little bit of binding for a wh‑‑ a while.
So continuing with buffered thiol, on the following page. But here dithiodiglycol, deionized and centrifuge‑‑ is available from earlier tests. So the experiment was set up with varying amounts of dithiodiglycol and a [00:31:00] constant amount of mercaptoethanol. And then haptoglobin was added. So here is a concentration effect of dithiodiglycol, control 2‑2 being completely as usual. But in the presence of 0.3‑molar dithiodiglycol, there are no polymers. And even at 0.003-molar, there are no polymers. But there’s a very good monomer. And so, in the presence of dithiodiglycol, mercaptoethanol produces a good monomer. The beginning of understanding, at last, although not yet written down. So this is page 109, Friday, October 18th, [00:32:00] that the best ratio is about 0.03-molar dithiodiglycol, with mercaptoethanol being constant at the usual concentration, which is not recorded, though could be calculated. So dithiodiglycol plus mercaptoethanol gives very good monomers. And the monomers bind hemoglobin and give a positive benzidine test, from the look of it. May not be a benzidine. It may just be a protein, at this stage. I should not say benzidine. [00:33:00] But that is a very important page, Fri‑‑ from my understanding, Friday, October 18th, page 109. At it becomes the basis of my later paper on resynthesizing. (pause)
Yeah. Here is a rerun, on page 3‑‑ 111, Monday, October 21st, the rerun of the Factor B tests, which is dithiodiglycol plus 0.03-molar mercaptoethanol plus about 1‑micromolar Factor B, and then varying the amount of [00:34:00] dithiodiglycol. And looking at these gels, the results show clear synthesis. That’s what I’m claiming. Because these samples are — were previously monomers and now are back to polymers. So that the 0.03-molar one, for example — I wan‑‑ is almost got back to… Well, it looks rather like haptoglobin 2‑1, modified. It’s partial synthesis of polymers. But polymers are really, clearly being remade now, when [00:35:00] a sample is allowed to oxidize. And so the remark is, “The results show clear resynthesis, but 2‑1-like, not 2‑2 and better in the presence of only a small amount of the dithiodiglycol.” “So test the [kaolin’s?] reagent,” is my next comment. But that, if — in fact, goes on as a retrograde step, as I would now know.
So — b‑‑ however, the… October 22nd, Tuesday, 113, 1,4-dithio-2,3-dihydroxybutane tests. That’s DTE, and dithioerythritol, and DTT, [00:36:00] dithiothreitol, available in cis and trans forms, etc. “Make a test with the trans form fir‑‑” And so I tested with DTT. “The results show splitting, partial, even at the lowest concentration, and no visible sign of wreckage.” So it’s a nice result, that you can get very good monomers — dithiodiglycol — at concentrations of — that are really quite low — though they — the best experiment is with point-oh‑‑ the highest concentration tested is 0.01-molar dithiodiglycol. And point‑‑ even 0.05-molar [00:37:00] dithiodiglycol — dithiothreitol, rather, DTT, the concentration 0.005-molar gives very good bands. And so the comment is, “The results show splitting, partial, even at the lowest concentration, and no — sign — no visible sign of wreckage. However, note that the hemoglobin appears to be partially wrecked, at the higher concentration.” And you can’t see the hemoglobin band at all. Rerun of the Factor B samples, treated — fro‑‑ page 113, being rerun on page 117, [00:38:00] really no — with the comment, “The resynthesis is no better. It could be worse,” the mercaptoethanol. But it’s quite clear with 0.01-molar. So I am getting some sort of resynthesis of the polymer. (pause)
Still thinking about Factor B and the oxidation, Thursday, October 24. “Effect of Factor B on oxidation, with mercaptoethanol and DTT,” dithiothreitol. [00:39:00] (pause) Resynthesize is being — rerunning sample again, Friday, October 25th, page 123, rerun of 0.03 SH plus 0.003 SS tests, at p‑‑ at 37, pH 7, 24 hours and 96 hour, resynthesis. With the comment that… [00:40:00] This is samples in the presence of hemoglobin, from the look of it. Yes, it says, “8:20 a.m. Samples out, and to hemoglobin.” And, “8:50 a.m. Back to rotation.” And then taken at different samples, 24 hours and 96 hours — and oxidation — and that the results say that “All have clear hemoglobin color,” at 9:15 a.m. That’s the — after 24 hours. And Factor B is very clearly catalytic, as Peel describes. And, “The plus-mercaptoethanol [00:41:00] sample is improved greatly.” So a comment on the preceding page, 132, “Resynthesis in plus-mercaptoethanol, plus-Factor B is quite good but seems to have stopped or at least slowed considerably.” And but DTT is put at 0.03-molar. Further refinements being tested, on the following page — and so on — the same sorts of experiments, Factor B concentration effect. No effect in going [00:42:00] from — going to 1-micromolar down to 1,000‑‑ up to 1,000-micromolar.
A wider pH range and temperature tests, on Tuesday, October 29. pH tests continued, pH, at 37°, Wednesday, October 30th. [00:43:00] Again, the resynthesis appears to be making something rather like haptoglobin 2‑1, rather than haptoglobin 2‑2. But no particular comment on this rerun test on page 131. (pause) Samples again, on October 31st, and looking at the ch‑‑ effect of pH 6, 7, 8, and 9. [00:44:00] And not very clear that there is any difference. “Initial pattern looks very good for the lower pHs. Therefore, test even lower.” pH 6 is a nice dissociation pattern. Starting material pH 6 is actually better than the pH 9, after 16 hours, which is what that comment is about. “Initial pattern looks very good for — lower pHs. Therefore, test even lower.” And with a result on the following page, “The results show pH 4.3 and 5 are essentially the same. pH 6 has not changed much, not different from pH 5. Therefore, try pH –” no — “try pH 6 at lower [00:45:00] temperature.” Looking at it from this point of view, there’s not much difference between the samples. pH tests after 20 days, on page 137, pH 4.3, 5, 6, and 7 and 8, with the comment that, “No significant change since 20 hours.” So 20 days at those pHs. Though looking at it from here, I can see that the hemoglobin is — has been destroyed at the lower pHs, 5 and 4.3. But not commented on in the book. Continuing with pH tests, November s‑‑ 4. [00:46:00] Continuing with varying various permutations of pH and temperature. Ionic strength tests, on November 7th, Thursday, page 147. “Increasing the ionic strength does not help,” is the conclusion.
And a general review on Friday, November 8th. And, “The general results of pH and plus or minus Factor B can be interpreted as [00:47:00] indicating that slow oxidation in the presence of mercaptoethanol is the best method.” (pause) It’s… So went back and did a test again, of air-oxidized material, made from different — in the presence of different concentrations of mercaptoethanol — showing reoxidation of the 0.002-molar was the best versus 0.02-molar, where no resynthesis occurred. [00:48:00] And so we’re getting to the end of this book, with a final test being recorded on about November 11th, on pages 152 and so on. And that comes to the end of this book. Yeah. [00:48:37]