So this is a physical book, Roman 10X, 1956 October, and it runs through January 1957. It’s a book that helps resolve many of the uncertainties of the previous books, because now I have good photographic documentation of what is happening. First two pages, just some families, nothing very striking. And then on Wednesday, October 31st, through Thursday, November 1st, a two-dimensional electrophoresis on the Douglas sample, the one that seemed to have an extra band for two-dimensional. [00:01:00] On the two-dimensional at 11:00 a.m., with a small sample of Douglas [whole?] serum, and off at 4:45 p.m., with a very good result. Shows that the [exisone?] is a slightly slow beta. And so the two-dimensional electrophoresis, with a photograph, makes it clear now that there is what proves to be a heterozygous sample with two different — again, later shown to be transferrins. It’s a rather good page, this page, early in this book. Good separations. The filter papers are shown, and the two-dimensional image is a photographic image. It’s not a very good photographic image, but perfectly clear and resolved. [00:02:00]
Some problems with slow alpha, too, November 1st. Not very critical.
Family on Thursday. There, the first…
Some auto entries, and then on Friday, November 2nd, this test is using an old gel, etc., etc., and I’m having images of the gel, which is a good step forward. Such an event, November 3rd, using KB17 film, photograph [varies?], nothing. And also, photograph of the two-dimensional gel on the Douglas sample. [00:03:00] Another family on November 5, and on November 6, happily, again, to see photographic images. Looking — and evidently I’d received a copper binding protein, the, the — from… Yeah, I’ll have to check who this sample was for — from whom the copper binding protein was obtained.
On Tuesday, November 6th, I ran a gel to see where the copper binding protein [00:04:00] — ceruloplasmin, as it was later called — would migrate, and also where an iron-binding protein migrated. I don’t know where I got these samples from. Probably from Dave [Pulig?], who used to get various samples, but this was beginning of trying to understand where the bands were from. But they — it was confusing, because the iron-binding protein didn’t migrate with any particular band, and I just didn’t understand the results yet, although later on I did. And the gel was developed with D-prime 11 — with D11, [00:05:00] I think developed for five minutes. It was quite a nice image. And next page is devoted to photography, again, re-photographed November 1st, big gel with a KB17 film, about 24 minutes, etc., etc. No improvement over them. And the two-dimensional one, trying different ways to improve it, but they were not too bad, as shown on the following pages, which show a photograph Tuesdays, experiments on KB17, using 14 minutes of development at A.E10 to bring out the contrast. But the contrast is insufficient, [00:06:00] but D11 with five minutes, etc., there’s some images of these same gels again, images of them with… Might as well get rid of the…
On Thursday, November 8, more gels, and Thursday, November 8th, re-exposing the two-dimensional one. And here I see some of the cattle samples, although it’s… Let me see what it says about that. Yeah. [They’ve tried?] more contrasting films for bovine in two-dimensional, but there is a bovine sample, as compared on the [00:07:00] left-hand page on November 8th, and shows multiple bands that we could see in those samples from Charlie Hickman. It’s a nice story about Charlie Hickman, because we went on and published about those samples, and worked out the genetics, all long distance from Ottawa, where he was. And one day a guy walked into the lab and said, “I don’t think you’ll know who I am.” A big, tall guy came in. And I said, “No, I don’t know who you are.” And he said, “Well, I’m Charlie Hickman.” And we had a delightful get-together, collaboration at a distance, and then eventual friendship in person.
Friday, November 9th, more gel photographs were of rather good images of both the cattle samples and the [00:08:00] two-dimensional gels. Still struggling, November 9th, re-photographing things. Unusually, the contrast rather too much, except for the faintest line, November 9th. More families, more families.
November 12th, [00:09:00] more gel photographs, another one quite nice. KB14 and D11 developer, same samples again, with the contrast only very little up from eight, some minutes with D11. This was 15 minutes. [Then we?] tried Kodak microfilm, which was five minutes of D11 gives a gamma of four, and a very high contrast film, which is the one that we eventually settled on.
Something University of Toronto, [00:10:00] some samples [of a vaccine?]. They’re not too very informative. (laughs) More films, November 13. This was Kodak microfilm, developed five minutes at D11, and a slight improvement over KB14, D11 15-minute, and yet only five minutes with D11. And they — the two-dimensional gel is really way better than previously, 5.6, number 24, two-dimensional, still could do with an [00:11:00] increase in contrast, but it’s very much improved — the gel is beginning to sparkle, you might say.
Some polio vaccines, tested with –- a comment that an unwise experiment. Color bands migrated at increasing rates from right to left, but I don’t know what they were, 42, 44, or 49. Some person whom I don’t recognize. CJ Smith recommends D85 for even greater gamma than D8, but with a need for a much longer exposure. But D85 is caustic, and hard on the motions. [00:12:00] And the contrast visually is less than with D11, so try D8, but ask Kodak first. You can contact Kodak. And check with Kodak. And they said that D85 is — gives a lower gamma contrast than D11, and that D11 will give 4.75 as the gamma, and they recommend D8 for increasing gamma more.
The gels really are looking very good by November 14th. Microfile, D8 for two minutes. The contrast looks [00:13:00] greater than or equal to D11. And a comment that (inaudible) gel was used, “ferricyanide if I have [too high a density negative?],” a November 14th comment. The November 14th, Wednesday, contrast’s best yet, even including Mr. Smith’s large negative result. He was evidently the photographer, because it was the result used for the Nature paper.
And so more images, two pages on, a whole bunch of images, obviously improved over earlier ones, and have a big red entry, D8, for three minutes [00:14:00] in the future. Settle for D8 at three minutes, with microfile. And that’s basically how we got to using that type of film. An interesting comment that a possible binding of aluminum three positives, MDP — that’s Dave Pulig — had noticed the effects of an aluminum dish on staining. And so a little bit of attempts to improve things with aluminum chloride on the next few pages. [00:15:00] With a comment that by morning the best was serum plus concentrated dye, without aluminum three positive, so it looks like ATCA breakdown is the chief stain in these experiments, whatever ATCA is. ATCA. Aqueous… Aqueous trichloroacetic acid, perhaps. Not clear, and not very important.
More families, [00:16:00] more samples from Negroes.
Friday, November 16th, tidying up old two-dimensionals, came across gels, which were half dried-out, and the increasing clarity was noticeable. In other words, you could get — the gels became transparent when they were dried out, and this later was something I tried to do, was to make the gels transparent so that I could get better staining all the way through the gels, rather than surface staining. It worked, but it was laborious. But this is the beginning of that sort of… This dry gel was steamed to flatten it out, and used as a negative, and you could see quite well what was happening. [00:17:00] But he comments that 50% glycerol, 50% [methanol gels?] do not regularly become transparent, so need to get the refractive index properly. Benzyl alcohol, one of the highest refractive index, could be tried, and I know, in fact, that it does work, under the right circumstances.
More families, and checks. November 19th, [negro?] samples plus checks. Titration… adding hemoglobin [00:18:00] in different amounts to Otto’s serum.
Tuesday, November 20th, two-dimensional. More tests. Then here, on November 21st, trying benzyl alcohol [00:19:00] for experiments and tests. And looking at the distortion. And the distortion was very great at 100% benzyl alcohol, but it was 100% transparent, no sign of loss of dye. And so I, I have a comment that the distortion, when it’s 75% benzyl alcohol, there’s only just a little distortion, and the transparency is about 90 to 100% of the best, and this is very good. Pliability and shape is excellent, etc. But I never really used this [00:20:00] much in practice, continuing with that idea. An alternative procedure will be to dehydrate the gels further by using higher methanol in the original wash before benzyl alcohol treatment, so that no further shrinkage need occur. Lots more samples, transparency tests continued.
November 22nd, [which?] were continued. And (inaudible) was a great deal of attention, but it was something I was interested in but never really followed up in any [00:21:00] significant way.
November 22nd, titrating the serum by adding different amounts of hemoglobin to get a sample, a Negro sample from wood fully saturated. I think at this point I still didn’t know this protein was haptoglobin. Transferrins tests continue. [00:22:00] Trying to get better results in the first dimension of the photo paper electrophoresis for two dimensions on November 23rd with different amounts of sample five microliters up to 10 microliters. Some new method of trying to insert the samples. Not very important. Results disappointing.
Saturday, November 24th, now looking at the buffer to use for the first dimension, the filter paper electrophoresis. [00:23:00] An XXM buffer was a buffer used at that time, and evidently the one that I liked best. Transparency tests continuing on November 26th. Trying some mixtures of benzyl alcohol and glycerol. Two-dimensional gels, again, November 28. [00:24:00] But no photographs. November 27th, some transparency tests, now with images made from them in very high contrast, contact gel, November 27th, which is a clear — that means transparency — made two-dimensional sample, direct print onto F4 paper. (laughs) Useless. The paper is too blue-sensitive. Tried different things. A contact print, 30 seconds, 17 seconds, etc., etc. [00:25:00] Dust must be avoided, but the [present?] gel is much faded anyway, but a fairly high con– very high contrast images. But too laborious to be worthwhile. It was interesting to see whether one could type the samples on filter paper without doing a gel electrophoresis, and so 10 samples were given to MDP Dave Pulig for group trials on filter paper, and all but one were correctly typed. [00:26:00] The poor one probably was low haptoglobin, as it was typed one instead of 2A, but that’s interesting that they could be typed on filter paper, that the change in migration was enough to see on filter paper. But it was never used.
Friday, November 30th, 10 unknown ones were sent to him for — to Dave — for filter papers try, and then tested later by starch gel, and fairly high success rate. About two out of ten were wrong, but 80% could be typed by filter paper. [00:27:00] (pause)
Looking at some samples from individuals who had had severe reactions on transfusion. One person here [00:28:00] was — it was commented this woman had serious cross agglutination reactions with all tested donor bloods. The serum clumped the cells, and (inaudible) maybe colder glutenins, but she had cancer of the bowel, and transfusion data says further blood to be given. I really didn’t ever do much work on pathological samples. I think I left that to Dave Pulig, who liked that, and other people. Some different filter paper tests. Five MM papers, an [S likely to show?] 598YDMB papers. They — I remember the names, but not [00:29:00] what were their properties particularly. Back to transparency tests on December 3rd, again, with very high contrast images obtained with the transparent gels. Trying different buffers for the filter paper on December 4th. More families coming. Still going back to transparency tests. Trying to improve the [00:30:00] filter paper electrophoresis on December 5th and 6th.
New dyes. I have new dyes being tested — [azicarbon?], [benzo phos?] red, brilliant blue, [sibilant?] scarlet, brilliant yellow, etc. — to find something better than the amido black, with a comment that brilliant blue, red, and scarlet blue are worth tests. [00:31:00]
Back on Thursday, December 15th, more gel photographs of various type. None of them look very good (laughs) at this point. [Which were artists’ writing?]? Clearly the red filter and the amido black gives the greatest density, and developing D8 for three minutes at 20 degrees centigrade, various filters. [00:32:00] [Tris-citrate?] borate gels were tested — they’re not very clear — on Saturday, December 15th, (inaudible) Tuesday. Yeah, and it does comment on Tuesday, December 18th, that the Saturday experiment was not well conceived. Aimed to see if tris-citrate will decrease the absorption by binding calcium ions possibly, so a tris-citrate gel was tried. [00:33:00] (laughs) Some comments. I remember vaguely hearing about this when I was a student in — where was I now? This was probably back in the days later… Let’s try again. Tuesday, December 18th, there’s a comment, all in red crayon, that a methyl thiol, CH3SH, excretion following the ingestion of asparagus is thought to be controlled by a dominant gene, [Alison Macata?], Nature. And we tested it on that day, and I know later on it turned out to be not a genetic problem [00:34:00] of excretion, but a genetic problem of the ability to smell the product.
More checks on samples, large number of samples, December 19th. And here, several more pages of checks by [Otto?]. Some tests starts again, different starches on January the 3rd. [Bovine?] serum received on January the 3rd from Ottawa, and were checked with the rather large gel, and typed into the types that later we understood one, two, three, four, and a 2A. [00:35:00] And bovine check on November 4th. Unfortunately, no photographs, but very clear agreement with previous data, the checkmarks showing that the types came out correctly.
Getting to the end of this book, another [name redacted] family with Otto running the gel. And here’s a nice little comment, January 7th, written in hand from Charlie Hickman: “Oliver, we had an accident with the enclosed something, something [00:36:00] blood. After receiving it in good condition from Charlestown, (inaudible) was put in the freezing compartment rather than in the refrigerator, and so everything was frozen and [smudged severely?]. I don’t know whether it’s any good, so I’m sending it to you anyhow.” And so these were a large number of very hemolyzed blood. Presumably this is a sample on January 7th of the material. It is received on January the 7th, the bovine sample, with a note from Charlie Hickman, and run on January 7th, [00:37:00] and with a comment that “Hemoglobin contamination shows in all samples the very hemoglobin of 15…” So, of doubtful use. Although, then, a summary of the latest bovines is on January 8th, and a nice little summary of the — of the results of one sample, though, with one sample of 5315 already typed as three, and it shows [00:38:00] new names. The names have been changed, so the new name and an old name. Deciding what to call the sample. I think rather than anything significant, because there are checkmarks against all of them showing that nothing is unexpected, but it shows a new name and a number, and presumably the old name. So one became three, and three became four, and it’s a little confusing, but solved in the end. The two-dimensional gel here. [00:39:00] Showing that if three hemoglobin have to go on a two-dimensional gel, separated in both dimensions, they separated in the filter paper dimension, the hemoglobin/haptoglobin migrated faster than the free hemoglobin on the filter paper, and yet, in the gel, the free hemoglobin migrated faster than the hemoglobin haptoglobin. So the two systems were — are different.
And the book is almost ending, with the latest bovine pedigrees, offspring, sire and dam. [00:40:00] So I’m beginning to work out the inheritance, with a little comment that 5202 sire was concurrent with 5205. Possibly the mistakes occurred at that stage of the breeding, because one sample did not fit into our hypothesis, 5202 and 5205. Everything fit our hypothesis as to the inheritance of these patterns. In the paper that was published on them, with Charlie Hickman and me, there’s quite a long discussion about that particular mistake, but there really isn’t any doubt that the [00:41:00] pattern of inheritance that we worked out was correct. And the last two pages of the book are hemoglobin checks on a whole bunch of samples, 2A, 2B, and 1, and on the left some charges for milling a plastic block, I don’t know what for. And that’s the end of this book.
Some clarification is required on book 10, fairly early entry, Thursday, November 1st, where several samples of purified proteins were compared with serum, and the proteins almost certainly came from Henry Kunkel at Rockefeller Institute, as it was called at that time, although they were not obtained by me. They were probably obtained by Dave Poulik. At least, I don’t remember obtaining them. And the results are fairly confusing, but anyway, let me go over them.
There was a purified sample of slow alpha-2, which is a [00:01:00] alpha-2 macroglobulin. The image of the gel doesn’t show this, but it says that the — it was equivalent to an aged slow alpha-2. In other words, they said it was slightly degraded or whatever, and not much doubt that the alpha-2 macroglobulin and slow alpha-2 were the same. But that’s not in the image. What is in the image is a comparison with purified albumin, which agrees with the albumin in the serum, plus some slower component, but mainly albumin. The sample of copper branding protein, 1%, is a sample of ceruloplasmin, [00:02:00] and doesn’t line up with anything that I could understand. And the iron-banding protein, which is transferrin, again, doesn’t line up with any band, although I now know that it should line up with what we used to call betaglobin. And this is probably because the sample from Henry was free from iron, rather than fully saturated. It’s a rather complicated story, because most of our samples behaved in the gel — that’s to say, most of our transferrin samples in plasma behaved as if they were saturated with iron, even though transferrin in normal plasma is not fully saturated with iron. That’s probably [00:03:00] because the starch used in all our work actually contained enough iron to saturate the transferrin, although I do comment in some other places in the journals that the pattern was a little smeary with the cattle transferrins, suggesting that the samples weren’t always fully saturated with iron. So it’s a little complicated.
A comment on the [couple?] binding protein there from Henry. That was a protein that — ceruloplasmin — was a protein which was associated, or the lack of which was associated with Wilson’s disease. And Alex — [00:04:00] Alexander, that is — Alex G. Bearn, B-E-A-R-N, was a world leader in studying Wilson’s disease, and he was also — I knew him very well, and enjoyed him, but he was probably the most articulate individual I’ve ever encountered. I remember at a meeting, a Cold Spring Harbor meeting concerned with genetic variations of protein, he had to summarize the meeting, and his talk was so beautifully polished that it sounded as if it had been prepared after several months of hard work, and it was just done off the cuff. He was one of the most articulate persons I know. He died a little while ago. He was two years older than me, born in 1923, [00:05:00] and he lived to be 86, so he had a good, long life, although, in this respect, I beat him. (laughter) Henry Kunkel, who was very well-known, again, from Rockefeller Institute, he received an award for his work on proteins. I’ll pause a moment to remember the name. (break in audio) He received a Lasker Award for his work on purifying proteins in plasma, and particularly, I believe, the methyl-binding proteins. A great scientist, and I use his method… Well, trying to use his method, Kunkel and Slater, in an earlier [00:06:00] book led to the invention of starch gel electrophoresis. So that’s the addendum to book ten.