And this is book R beginning January of 1965 and continuing up to almost the end of 1965. This book has some references to earlier ideas as I will try to make clear. The beginning is just continuing the haptoglobin 2 splitting tests and so on by running two-dimensional gels. Notice this is Hp2, not Hp2-2. Hp2 meaning the monomer. Hp2 monomer, etc. And continuing to investigate that situation in two-dimensional gels in the following pages.[00:01:00] Continuing to try with urea on Wednesday January 20th page five at room temperature. And so on. And next few pages. Haptoglobin 2-1 monomer in quotation marks electrophoresis on page 11. Rather poor-looking gel. Page 12 and 13 April 8th, 1965 written in presumably [Nancy’s?] handwriting. It’s talking about [00:02:00] [Dylan normal red cross?] borate serum. Trying some cross-polymerized beads Wednesday May 5th page 15. And some haptoglobin final, these are presumably ready for publication, that I’m trying to get images in the different stages. So page 18 has a gel that’s [00:03:00] pretty close to a final type gel of haptoglobin split and run in 8 molar urea. After splitting in 8 molar urea and borate and with mercaptoethanol and then run in a formic acid gel. This looks very close to a final publication gel which we identified later. So then there’s page 18 and page 19 of book R getting close to the images used in describing what happens in the haptoglobin system, quite nice gels. [00:04:00] So again trying to get good images for publication.
Haptoglobin monomer resynthesis on July 15th. Effect of mercaptoethanol on resynthesis. Beginning to understand protein SSMe in the presence of mercaptoethanol S- goes to protein SS protein plus MeSSMe. I mean it’s quite clear that I now am understanding what the resynthesis mechanism is. So [00:05:00] this is Monday July 19th page 29. Clear statement of what should happen.
And tests on the following page. First of all the reaction after 19 hours’ storage at room temperature and then at 33 hours. And showing monomer control and then the effect of the monomer plus mercaptoethanol getting partial resynthesis. In fact the resynthesis looks like [00:06:00] making something rather like 2-1. No significant change with time but obviously resynthesis is occurring.
Thirty-seven-degree test of the resynthesis on page 35. Trying to see if that makes any difference. No comment. Quite apparent that there isn’t really any big effect. [00:07:00] Page 37 actually is the beginning of the answer to the resynthesis too. Although not obvious yet. It just says on page 37 note, once the best conditions are established, increase the concentration of haptoglobin. Which I know from the final result turns out to be very important.
Page 39 final gel of haptoglobin 2-2 resynthesis. Doesn’t look terribly impressive. Beginning to have some thoughts on illustrating this reaction. It’s apparent on Monday August 2nd. I know became part of the [00:08:00] final paper on the use of [dithio glycol?] and reversing things. Is coupling of glutathione to hemoglobin [knowing?] the presence of an SH group. So this is testing hemoglobin to see if I could alter the mobility of hemoglobin by combining with haptoglobin. The reaction is fast and can test with half an hour at room temperature, etc., etc. The result is not very clean in this particular test. But it’s the beginning of what later turned out to be quite good.
Repeat of the GSSG test. That’s glutathione disulfide test with [00:09:00] new hemoglobin on page 43. Set up what to expect. And the result shown in a gel. The change in mobility is good to see. That’s really not a correct statement. The result on page 45 is presented as what happens with iodoacetamide and glutathione and iodoacetamide alone, etc. [00:10:00] The benzidine gave trouble. The gel actually looks quite nice but not satisfied with it.
Higher pH on the next page. And testing again the effect of glutathione disulfide on the mobility of hemoglobin. So test also with cystamine. That would introduce a positive charge. Would be fairly easy to see. Thirty-seven-degree for glutathione disulfide and cystamine test. Which is [00:11:00] cystamine dichloride is actually [NH3+, CH2CH2SSCH2CH2SNH3+?] cystamine. It’s amino instead of [dithiodiglycol?] it’s [diaminothiodiglycol?] I guess. But anyway it introduces the positive charge.
With a comment that clearly it’s on the borderline for severity as judging by [A2?] but GSSG is more [extensive?]. There’s clearly effects there. [00:12:00] On Friday August 6th, repeat with the cystamine result of — with some very clear gels. Which are probably actually used in the paper as we will later see. [00:13:00] So I’ll stop for a moment and check the paper gel. So the gel on page 50 run on Friday August 6th is used in my later paper on disulfide-bond cleavage and formation in proteins which was published in December of 1965 in Science magazine. Quite nice paper. In fact looking at it on Google I find that it’s been quoted 92 times. And people were using it as designed, to control the resynthesis and cleavage. The cleavage and resynthesis. Of thiol. Of disulfide bonds in proteins under controlled conditions. So the gel on page 50 is [00:14:00] Figure 3 of this paper.
Look back to see which gel was used for Figure 1 in that paper. You see I’ve identified the gel that was used for Figure 1 Panel A. This was run on Saturday December 12th, page 88, top left-hand gel. Although the photograph is rather faint, I’m pretty sure that that’s the image which was used for Panel A of Figure 1 of the disulfide paper.
I don’t know whether this is now a correction or not. But the Panel B of the [00:15:00] paper in Science was on page 88 Saturday December 12th. I think I’ve already commented on that. It’s Panel A is a little bit more difficult to find.
The two figures, the two panels, in the Science volume 150 19 whatever it was, 1965, paper are page 82 and page 88. The figures are there taken from those two pages.
So we continue. [00:16:00] With having identified basically the figures and the ideas behind the disulfide paper. So continuing then with book R. Some digression. Testing bisulfite reactions also of the sort that Rupert Cecil had done. In the following pages. And also tests with nitrobenzoic acid disulfide, which gave an acidic product. And tested it with hemoglobin and got the expected result.[00:17:00] OK, this reagent will work, some degradation product, etc., etc. The little arrow. But basically it worked on Sunday September 5th. Now on the following page, 58 and 59, Thursday September 8th, is a complete change of thought. And here are beginning experiments I think probably with Alice Claflin looking to see whether antibody cells could divide. Exactly what the motivation is is not clear in the book although I think it goes back to a paper on antibody [00:18:00] virus which I had a paper I don’t really think was worth anything. But it was published as a theoretical or hypothesis paper as we will see. This was a paper published in July of 1965. And so this series of experiments carried out in September of 1965 are beginning of some tests intended to test this hypothesis. In an earlier book. This is the beginning of tests related to a general plan for antibody [00:19:00] transfer tests, which I had already pointed out in book Q page 13, a general plan for antibody transfer tests. The idea being to take litters of newborn or prenatal cow cells, allow them to grow, and ditto with sheep cells, newborn or prenatal, and immunize them if possible with different antigens, and then see if one could crisscross things. So the idea was cow cells. Allow them to grow. Sheep cells. Allow them to grow. And continue injections from time to time to see whether it was possible to transfer the antibody producing [00:20:00] capability from one cell to another, which was what was being discussed in this rather in my mind at this point rather silly paper on antibody induction and tolerance which I had published as I said in July. Anyway these tests in book R are towards that idea. And was done by radioactive tests where tritiated thymidine was — cells dividing or cells being cultured were exposed to tritiated thymidine and the idea being to look and see if any of the plaques had incorporated DNA or thymidine [00:21:00] indicating that they divide. So there’s a long list of what the various cells looked like on page 58 from the autoradiographs. And continuing on page 60 and 61 with some on page 61, summary. Thick areas lead to diffuse plaques and dangerous label. Only superficial cells are safe. Therefore count only thin slides as far as getting the autoradiography to match the microscopic images. Going on Friday September 9th. Many sketches of what was happening in these cells. [00:22:00] But here and there cannot see the cell. No label. Clear but small plaque, etc. So I’m looking for plaques and looking for incorporation at the same time. Which later became part of or in fact the bulk of Alice Claflin’s 1970 thesis on antibody cells in division.
Continuing with several more pages. And a number of grains of autoradiographic tracks being recorded. For example on 66 a large clear cell with 50 grains over it. Indicating that it had probably divided already. [00:23:00] Or had incorporated thymidine.
Two hundred microcuries used on page 69 September 12th. Here and there junk, zero, junk. No label. So not very happy, following pages much the same. Her trying to do a different method and trying to get imprints of the cells rather than trying to look at the gel directly on page 73. And double plaques on Tuesday September 14th in animals. Six animals were given one and two and three and four, [00:24:00] which were animals one and two were given 50% cow cell, three and four were given 50% pig cell, and five and six were a mixture of the two cell. Aim to plate anticow, antipig, and antipig cow on cow pig 50-50 mixture, etc. And looking for clear plaques. Trying to see what the antibodies were.
Back to making Bence-Jones proteins or checking on them on page 79 September 21st and some rather nice gels on the following page. I must have been rather happy to have that break of gels. Trying [00:25:00] SSR reaction with the Bence-Jones protein on September 23rd. And saying the results are quite happy of [Nevin?] multiple myeloma versus — oh, that’s a different experiment. This was [Nevin’s?] multiple myeloma serum I think versus the Bence-Jones protein. Which of course was what eventually Gerry Edelman with Poulik showed using the 8 molar urea in this case with [00:26:00] barium lactate gel.
And more tests with [Nevin?] on page 87. Then returning again to the cultured cells on Tuesday September 28th. Pages of sketches. Two pages of sketches of the cells and so on labeled background. Random label, etc. No label. No label. Eight grains, 12 grains over [all?] cells, etc. Not pleasing I’m sure at the time. One grain, no label, no label, page 90, etc.
Average about five. Therefore the above [00:27:00] results are probably by chance. Attempting to improve the situation by taking imprints. On page 95 with a big square saying background rather high, do not use these data.
Yes. Here there’s a very clear notation that it’s Alice Claflin because there’s a note on page 99 saying separate check by AC on [spleens?] shows one in four were high. So response is there. Meaning we were getting antibody. But it’s Alice Claflin.[00:28:00] Continuing these experiments over the next several pages. With some of the writing being by Alice and some by me. And then pleasing result on page 109, Tuesday October 19th, 1965 in Alice’s writing. And then in my writing at the bottom. One of the dry photos has a metaphase. [00:29:00] Animal 22 has a metaphase at the position being 112.8 by 5.6 on the microscope stage. Continuing the work in the following pages.
Struggling with the technique. Then on page 117 some imprints taken with light microscopy at high power. [00:30:00] Mounted in immersion oil under the cover slip and some rather beautiful images of clusters of cells with a comment on the left-hand side that this plate was nearly ideal, about six hours dry, etc., etc. Stained with [sergeant?] Giemsa and so one can see the morphology of the cells. Page 119 cell there which says this cell is a monocyte and this cell is a dendritic macrophage. So cells [00:31:00] beginning to be recognizable with a high power lens 1.25 with new camera and no antishake precautions. Page 125 is a whole series of exposures taken with Polaroid film. There’s a cell. Looks as if it was a metaphase cell from looking at it now. [00:32:00] The one shown in the top left-hand on page 120. And continuing with these experiments with Alice. Looking for cross-reactivity between the antipig and the anticow serum four days after an immunization. So these will be IgM-producing cells. Producing — will be IgM antibodies presumably. And the cross-reactivity is [00:33:00] quoted on page 130 as less than 5% but greater than 2% of pig versus cow and cow versus pig.
This was assayed by the number of plaques obtained with the cow cell. With the antibody cells being plated into agar that contained the red cell. So the clear plaques are where a cell is producing a hemolytic antibody. So for example 36 out of 5,130 were clear [00:34:00] of the number of total plaques, 0.7%, etc. Average being 3.6, page 130, looking at the antibody — the cross-reaction by counting plaques that hemolyzed either all of the cells of one type and none of the other or hemolyzed partly both types of cells, etc. Quite good experiment.
Several pages following all in Alice’s writing. And she wrote down what the gel was on page 140, 141. So continuing in the same vein. The [00:35:00] rest of this book is all in her handwriting. And it ends on Friday November 26th, 1965. End of book R.