Oliver Smithies:
[00:00:00] So here we come to Book C, starting in 1960, and it really is a continuation of purification, and so on, type of experiments. Clotted plasma, for example, beginning on page 1, August 10, with the eluted material, and seeing that the clotted plasma, which of course is serum, the serum is essentially equivalent to plasma, but pH 4.3 is appreciably poorer than 4.6, so here is serum versus plasma comparison on page 3. Zinc adsorption tested with zinc acetate [00:01:00] on Thursday, August 10th, the zinc experiment. Nothing remarkable. Serum versus plasma test again on page 9, August 11th. And, a comment as previously, the hemoglobin-haptoglobin is greater in plasma than in serum, but otherwise no significant differences.Two-dimensional splitting, I don’t know what that was particularly. [00:02:00] But the gel wasn’t a good gel. More purifications going on. Electrolytic reduction here again, the idea, obtained 12V auto battery, charged overnight until [gassing?] 1-2 amps, and then open circuit about 12.8V, and allowed it to settle down, etc., and test of stability of the voltage, why I was wanting to do this, it’s not clear. Though, comments on being perfectly adequate [00:03:00] control.
New fast column, August 16, looks fairly reasonable. The haptoglobin and impurities are well-separated, but too spread out is the comment of this column. Testing splitting haptoglobin 2-1, two-dimensional this time again, so running 2-2 in one gel, and 2-1 in another gel, and then right angle’s the formic acid, 8M urea. [00:04:00] There’s a sketch of the result on the 2-1 material, but no images yet.
More purification. Two-dimensional gels results shown on August 21st, Sunday, page 37. Looking at them now, they look rather poor. No particular comment. More images on page 39 [00:05:00] with the comment that there’s a — very good but low yield, sent the material to Gordon, that’s Gordon Dixon in Toronto, sending him material for —
This is a mistake here; this is August 22nd. Let me start that one again. Monday August 22nd, page 39 is cleaning up the haptoglobin 2-2 again, with a column to clean up the material to send to [00:06:00] Gordon Dixon, and evidently, quite satisfied with it, because I have the comment that it was very good, but low yield, and sent to Gordon Dixon, purified 2-2.
Still thinking about this electrolytic reduction with complicated diagram, trying to elute with a platinum electrode, and the reference electrode, etc., and stirrers – I don’t know why I was so interested in it, at this point it looks quite pointless, but at that time, I thought it was worthwhile. There’s a comment, for example, that on page 17, “All [00:07:00] show no effect. If unable to obtain reduction with platinum, return to mercury.”
So, the comment on Thursday is that, “Still all negative, but recheck,” and so tried again with a mercury electrode. With a comment on page 51, “Unsplit, partly split, almost completely split, completely split,” so you could get some reduction with a mercury electrode. So there are no gel images that I can see at this point. [00:08:00]
Still puzzling over it, test of haptoglobin 1-1 and haptoglobin 2-2 on electrolytic cleavage, and of the hemoglobin binding [blocked?]. Gel setup shown, but no images. Mercury reduction in the presence of hemoglobin. More resistant to cleavage in the presence of hemoglobin, but the poor quality hemoglobin makes binding uncertain, comment on hemoglobin, mercury reduction [00:09:00] in the presence of hemoglobin, Sunday August 28th.
More attempts without urea on page 61, all without change. No cleavage, page 63. Check if haptoglobin 1 versus haptoglobin 2, Hp1 versus Hp2, and hemoglobin binding. So these were materials that have been reduced in 8M urea, 0.2M ammonium acetate, and electrolytic reduction. [00:10:00]
(inaudible) obscure notes. And trying to work with my first wife, Lois — LK Smithies, Lois Kitze Smithies, and antigen, looking at a summary of separating it on a column. [00:11:00]
Trying para-aminobenzyl cellulose on Thursday September 29th, page 81, results show no haptoglobin removal, or at least not — (inaudible). Still negative on the following page. Looking at the resolution of haptoglobin 1F and 1S, that’s all, at this point, no, it’s clear that without any comment, that 1F and 1S are being considered, so I think I must at this stage have heard George Connell’s [00:12:00] results that 1F and 1S were, in fact, genetic variants, and not chemical variants. And so I go and see whatever I can find when each paper was — when I knew his paper, or if these results, this is page 85, September 20th.
The entry on Friday, September 20th, page 85 is a little bit confusing, because it’s talking about the urea concentration and the resolution of haptoglobin 1F, 1S, and 2, but whether at this point I knew that 1F and 1S were genetic differences is not clear. However, the result is [00:13:00] pretty obvious that 1M urea up to 3M urea, in formic acid gels didn’t resolve 1S, 1F, and 1S in any of them.
Then began to think about other things, Monday October 3rd, acid chloride coupling. [Isthica?] had used SOCl2, and XE-64 [or 93? carboxylic resins, etc., etc. So I refluxed 1g, and it’s not clear. [00:14:00] I’ll do that again. It’s (inaudible) — testing some — I’ll stop it, Jenny.
On page 87, Monday, October 3rd, I tried to modify the cellulose, carboxymethyl cellulose, using a method that [Isthica?] had described, using thionyl chloride, SOCL2, so I took carboxymethyl cellulose and refluxed it, in the presence of SOCl2, and with the solvent being chloroform to try to make a cellulose which could be used for preparing the haptoglobin, and comments on whatever’s happening. [00:15:00] After washing the material, I dried it. But, no sign of haptoglobin removal in using this material. And some comments on this column, on the following page.
Trying para-aminobenzoic acid, diazotization on October 4th, [00:16:00] with a comment on page 94, and negative again. (laughter)
Still thinking about using hemoglobin as a chelating agent to purify the haptoglobin, on Thursday October the 6th, come trying to make insoluble hemoglobin, possibly with making a gel of hemoglobin by cross-linking with an epoxy compound, or formaldehyde, or doubly-diazotized benzidine, and use it, so took some samples and tried to see what I could get. [00:17:00]
On page 99, some of these material was used, and some indication of haptoglobin removal, so to look and see if I could remove haptoglobin with this insoluble hemoglobin. Negative again pH on page 101. [00:18:00]
Page 107, October 18th, simple ionic cellulose hemoglobin test. I took Cellex-P, the phosphate, adjusted to pH 6 with ammonia, and said — tried to see if I could bind hemoglobin to —
Thinking about complicated double columns, page 109, [00:19:00] back to splitting again on page 111, alkalized splitting. In reviewing the work ready for publication, I noted the last result prior to using urea thioglycolic acid work, was splitting an alkali. So I used sodium hydroxide, a theoretical pH, about 2.4, for different lengths of time, tested in formic acid gels, and 8M urea formic acid gel, but no images.
The comment on Sunday, [00:20:00] October 23rd, the check of a result showed that haptoglobin 2-1 and 2-2 had never been split without urea, and run in formic acid gel, so I made up mercaptoethanol in borate buffer, added 2-1, and tested, and showed that they split normally. The usual result, haptoglobin 2 only, Hp2 only from 2, to Hp1 and 2 from 2-1. That’s mercaptoethanol and borate. Looks as if there was no [00:21:00] urea in this experiment.
Serum versus plasma, comments on splitting on the column, etc., (inaudible) [tested?], page 117, “Plasma is equivalent to serum, but repeat the electrophoresis for a check.” Rough test of trying to split on the column. [00:22:00]
Column splitting continued. Quite a nice looking gel on page 123 in relation to the column splitting continued. Heating the gel, the column in some way, let’s see whether I can make sense of it.
Going back to page 121 [00:23:00] “Since very little other than haptoglobin 2-1 came off on unheated column, after acetone precipitation, try the acetone precipitate.” So, 30% acetone, 10% mercaptoethanol, and 0.002M 2APA, etc. [00:24:00]
Anyway, the results are on page 123. Various degrees of heating; it doesn’t appear that the heat had any effect. There was a less — this is a formic acid, 8M urea formic acid gel, and it’s clear that the heating had removed the constant part of the haptoglobin product. It’s not — no comment on that, but it [00:25:00] was later, I know used as the method of purifying haptoglobin Hp1 and Hp1F and 1S, and Hp2, free from the constant region, by heating the material, but I don’t know whether I really realize yet that this would be helpful. Though I think some interpretation, though the heating is not clear what that was, page 123, it says that the result is understood, because approximately pure Hp1 and 2 in later times, excellent result, looks as if 10 and 20 minutes heating is good. But no decrease in Hp1 from 0 to 20 minutes, [00:26:00] probably 20 minutes is the safest, with a comment on the gel, which was an 8M urea formic acid gel that this 2-1 is Hp1 is Hp2, 1F. So beginning to understand a little bit about 1F and 1S, so the genetic differences, known, I think by this time.
Serum plasma test again. [00:27:00] Slightly a repeat of the columns, with the heating and the acetone, maybe makes more sense this time, Thursday, November 3rd, page 127, two columns of haptoglobin 2-1 were tested, 100mLs of acetone, and 70 volumes of acetone, 28 volumes of distilled water, and 1 volume of 2M Tris acetic acid, pH 7.5, etc. And, heating. Here is a little bit more information. 20 minutes at 70 degrees, 20 minutes at 85. Two of the comments, any of the heating ones showed, [00:28:00] and all had been heated for 20 minutes, and all of them show there is no constant region material in there, with the come to — all the split OK, in 1% mercaptoethanol Tris, and the elution is OK. And noticed another result that there’s no change in the fast or slow impurities on longer heating. Therefore, stick to a lower level of heat. I know where this is going to go, but it’s not yet clear that I knew at the time that it was going to give a method of purify Hp1F, and 1S, and Hp2, for typing very rapidly [00:29:00] without making complicated experiments.
For example, here we are on page 135, that the results show that pH 5 elution of unheated does remove much of the slow impurities, but it also removes most of Hp1, with (inaudible) — etc., etc., so some problem in that. [00:30:00]
Continuing with different variations. Nothing very helpful. #146 on Wednesday, November 16th, Hp2, Hp1S, underlines the 1S, so it’s clear that I do understand now that 1F and 1S are different things. The history of it is that basically, that George Connell discovered the difference because he began to make samples from individual donors, and then found that if he had a 1-1 individual, he might get 1F/1F [00:31:00] or 1F/1S, or 1S/1S, as it were, as we later called them.
The book ends with a test of splitting conditions, with this acetone method. And so ends book C.