Oliver Smithies:
[00:00:00] Book H, capital H, which starts in 1961 and goes to 1962, still, of course, at the University of Wisconsin. Another gel looking at the haptoglobin types, subtypes, versus insulin, with eight pretty gels, and many measurements on the following two pages. And continuing to do the same sort of work over the next several pages.Lysozyme and insulin on December 12th, page 9. And again, how people were generous in sending their material. This is from Franco [00:01:00] Conrad. “Enclosed, please find about 20mg of tobacco mosaic virus protein, lyophilized, et cetera.” And then there is a gel there with, showing how that protein [migrated?], or didn’t — with a comment that “TMV Protein, December 14, page 13, Franco Conrad’s gift, et cetera. And the rough test on filter paper, on FP, showed much more protein in TMV virus suspension than expected.” [00:02:00] There was a very pretty gel, and sample three, the third lane is the virus suspension. Her fourth lane is the purified protein. That’s a very nice gel.
Now, putting the TMV protein into the eight-gel tests, on the next several pages. [00:03:00] And some tests of hemoglobin, of haptoglobin versus haptoglobin plus hemoglobin 1-1 and 2-2, in the different starch concentrations. (laughter) With the usual comment on page 24, “Still very promising results.” And the calculations on the following pages. And further experiments of the same type in the next few days.
And then images and calculations on page 32 and 33, of the haptoglobins, different, this is a 2-2 haptoglobin [00:04:00] at different starch concentrations. Trying to determine whether the multiple bands fit some sort of regular equation for band number N versus molecular size.
On page 39, there is a gel, this is Thursday, January 25th. There is a gel, 8M urea formic acid mercaptoethanol gel, containing insulin B-chain [00:05:00] and the alpha and beta chains of haptoglobin in eight gels. And the image on that page, 38, 39, is what appears in the publication, unless I’m mistaken, you see. No, not quite yet, very similar to the publication, but not yet the same one. February 1st, Thursday, various odd haptoglobin, just being checked in regular borate gels. A sample from Copenhagen, [00:06:00] and from Oslo, et cetera, trying to type them.
And I noticed there that there’s a Polaroid image on page 46, they’re beginning to realize that we wouldn’t have to go on constantly developing film, so, and printing in the dark room that we could use, the Polaroid. The Polaroid image on page 46 is quite good for a beginning of that procedure, which later on, we use all the time, and beginning to use it already on page 53, although the result is not very good photographically. Still thinking about recent [00:07:00] cutting out the major leading band in the haptoglobin 2-2, and the preceding, rather fainter band that was always pleasant in 2-2 haptoglobin, try to cut the marker and seeing what would happen when treated in different ways, cut out the various bands and freeze and extract, et cetera.
Cut out, and another plane, doing two-dimensional experiments. Test of cutting with mercaptoethanol all weekend, page 66. [00:08:00]
A family sent to me by [Allan May, Dr. Allan May?], and Dr. [Krug?] who will be most grateful for an interesting, interested in the results of your determination of the transferrin and haptoglobin types in a family where one test was, whether the two persons were identical twins.
More Polaroids coming again, this time, some samples [00:09:00] for Lois Kitze-Smithies. A test within atoms of insulin electrophoresis, where they — yeah, 3.3mg of insulin on test, and also beef insulin, et cetera, and looking at the gel, the gel area is a regular photograph, [00:10:00] not immediately matched with the diagram opposite. It’s quite a nice little gel, (inaudible).
Some more samples, on page 89, I used to get samples from all over the place. This one was from Dr. CM Riley, University of Colorado Medical Center, Denver, Colorado. The plasma was thought to be, what was called the “Carlberg type.”
And a letter sent from [00:11:00] Kings College England to George from Betty Robson, who was a longtime associate of Harry Harris, if I remember correctly. But she’s writing to George, and I have the letter here.
“Dear George, after excavating through four years’ worth of material, I have found the remains of the sera from the Italian family showing the abnormal haptoglobin segregation. As I fear, there’s very little — I don’t know whether it would be of any use to you. But anyway, I’ll send it.” And so, I have the letter which went to George. [00:12:00] And I tested it following James Edwards, who was one of the individuals involved. And nice memories of Betty.
Preparation of — this is a different – there’s a mistake there about the (inaudible) one on page 93. That’s a bigger family, [00:13:00] bigger samples available, because it was purified on page 95.
Rather interesting comments on page 99, beginning to understand a little bit better what’s going on with this recent, “On looking up the chemistry, it’s doubtful if acetone acts as a hydrogen bond cleaver; rather as an equilibrium shifter, since the reaction following it is rapid at room temperature, so I wrote a diagram of the possibility that acetone reacts with mercaptoethanol to give a thioether, which is difficult to [00:14:00] reverse. So beware of resynthesis, but to use urea in concentrated solution and then dilute for resynthesis, or Sephadex as in the past.” So I was getting worried about the chemistry of acetone and mercaptoethanol.
Recent, this is cutting out various parts of the gels on page 103. “Must be able to prove resynthesis following cleavage without urea. Past attempts have not been successful, et cetera,” so still trying to get [00:15:00] resynthesis to work.
The purified [Carlberg?] from George being tested on Wednesday, May 2nd, page 109. Saying that here, the Carlberg, comparing the subtyping. It looks like a haptoglobin 2, doesn’t look any different at this point. But [00:16:00] there is something migrating very slowly behind the constant region, as if this is a variation in the constant region, rather than in the variable part. And the comment being that it confirms the subtyping, but a very slow component, post-beta, could be the other alpha chain is the thought.
Resynthesis again on the following day. Resynthesizing the presence of copper on page 115. And, the Carlberg molecular weight determination on 119, using the multiple gel concentrations. [00:17:00] With a comment on the images, “Quite good,” and then the comment, “Clearly, the questionable alpha chain has a very large retardation, greater than 2J, that shows marked asymmetry.”
This is a gel with Carlberg in formic acid, all the concentration, along with insulin B-chain. Now, at least, some more formal [00:18:00] test of Polaroid on page 123, full tests of type 55 P/N, positive/negative Polaroid film, with a test object. A range of prints are acceptable, with at the bottom, Polaroid 3000, tested at three best exposure, and at the same magnification. Micro-File, ditto, without and with Red-A filter. And then many photographs on the following page, regular photographs. I’ll look for the Polaroid one. When they’re trimmed, it’s not easy to see which are which. [00:19:00]
On page 125, there are 11 different photographs of Micro-File, plus or minus filters. And on page 127, the same gels, photographed with Polaroid 3000, comparison, with a conclusion that 55PN is the best for medium to high contrast objects; the negative is better than the original print. [00:20:00] But, clearly, the Polaroid is going to overtake the use of other methods.
Although, on page 129, test of Micro-File, Red-A, and D23 printed on F4, coated bromide paper, the Type 55PN, that’s the Polaroid one, is inferior to Micro-File for low-contrast material, but OK for routine, so very low-contrast material, it isn’t as good as (inaudible) Micro-File, not surprisingly, really. [00:21:00]
On page 131, June 5th, the separation of the 2-2 polymers being tried.
On page 133, different fractions of haptoglobin preparation, of haptoglobin 2-2, with a Parafilm Marathon stapled on page 132, maybe Parafilm had just become available, because of course it was used very extensively ever since, no comment on there.
Resynthesis, June 5th, page [00:22:00] 135, attempted. No signs of anything at work. Although on page 137, resynthesis continued. The comment, “The resynthesis is under way, but repeat with less hemoglobin, and with 2-1, and 2-2 controls,” so this is an attempt at resynthesis from page 135. [00:23:00]
Trying on the following page to improve the separations of haptoglobin to separate the different change with a 2% [alga?] column, that doesn’t look to be very promising.
On page 135, “Result shows clearly that splitting was incomplete.” So, there’s always the task of being sure that splitting is complete for having the resynthesis. [00:24:00] Trying to use Sephadex Pelikan Waterproof Indian ink, as an indicator for use on the gel. So one drop of Sephadex ink, and doesn’t have any obvious effect, not much in polymers, page 145, and [00:25:00] so we’re going towards the end of this book.
Acetone and urea resynthesis experiments, page 149. Still haven’t understood what’s going on. Repeating the tests on page 151, with a comment, “If unsuccessful, try 50% acetone.”
And the last entry in the book is, “Urea, pH range repeat run.” Really showing the dissociation, rather than the resynthesis. [00:26:00] And that’s the entry in this book.