This is a physical XVII, starts in April 1958, and runs through August of 1958.
It starts with a vertical gel, April 9 through 10, pretty vertical gel with two beta-globin variations, with a faster beta-globin, and the normal, or more common variant, both of course are normal, very clear. Another vertical gel. Vertical gels are now very common. This is a particularly pretty one, April 10th, 11th, with the post-albumins [00:01:00] showing very well. I must have been pleased with these results. One with the slower variant, now with the faster variant of beta-globin on this gel on April 15th.
More vertical gels, April 16, 17, very pretty haptoglobin patterns in this particular one, which was a check on previous results, with a comment that they check completely, on, there’s a 1, 2a, and a 2b, [00:02:00] quite spectacular results, even looking now from a later time, vertical gel.
April 28th, Thursday through Monday, April 28th, Thursday through Monday, attempts at non-slicing gels. [00:03:00] Not quite clear what is involved here, but it looks like three layers of gel, results fairly good. Comment that it would be good to have transmitted light to get rid of some of the troubles, messing around with different sample methods again, Sunday April 27th. [00:04:00] Some photographic tests will going on.
Vertical glass water-cooled 3mm gel was run on Wednesday, May 7th, with a comment that, note new resolutions in the post-betas, they are quite nice; I don’t know that I ever followed them up, but the results are quite pretty, and I’m clearly pleased with them. This is a vertical glass, and water-cooled 3mm gel. [00:05:00]
Looking at the post-betas again, a whole bunch of samples that were haptoglobin, type 1, presumably. There aren’t any haptoglobins visible. But these are samples, 3-5-2-9-5-7-3-8, etc., etc., probably from the Aborigines, but quite a lot of variation in the — they’re all haptoglobin 1-1, and they were chosen so that I could look at both betas. As it says, they are all haptoglobin 1-1s for [00:06:00] post-betas, and the 20 volts, [percent to media?] high voltage, and the result is very good, although a lot, number 108 of starch, should be even better. However, there are very clear variations in this region, and I never was able to follow all the variations that I could see. But that is a very pretty example; it looks almost as if there was a homozygote, a heterozygote, and another homozygote, variations in these post-beta sample.
Trying to get transparent — [00:07:00] — again, (inaudible) by Friday, May 9th, still using high voltages, and looking for various features. This particular sample says that all of these voltages, the zones are self-sharpening, and the post-albumins are very good, and post-betas are clear of all haptoglobins, even when there is hemoglobin present. Clearly, a lot of variation.
May 9th is somewhat interesting. There is a regular [00:08:00] photograph of this gel, which was run at 20 volts, [percent to media?] rather slow with water flows, with evidently a cool gel, and the resolution is really quite remarkable, and it says, of the post-albumins, perfect post-albumins. But alongside is a Polaroid image, perhaps not of the same gel, but it’s the beginning of the fact that we could take images with Polaroid, rather than have to develop all the images with [microfilm?] then and print them, it’s clearly a Polaroid, quite well preserved, considering the length of time. But there is no comment about this, just attached to the [00:09:00] page. Somewhat uncertain whether that attachment is a later attachment or not, because it’s stapled into the page, and no evidence that it had been fasten in with tape so I’m a little suspicious about the Polaroid image that is attached there, May 9th. The regular photograph was very clear, and very good images.
Monday May 12th, remade the water glass tray with efficient cooling to the edges, and it was [off?] a bit early. [00:10:00]
Something I call a dynamic equilibrium experiment, Tuesday, May 13th, run on a one-dimensional for eight hours. And then starch, sectioned and frozen, inserted into the gel, trying to get out the various components. So, it’s trying to be sure that there aren’t artifacts, and [it’s clear?] [Hurley?] said that our all three bands are as when originally prepared, so cutting out the three bands, these are the haptoglobin bands. Dr. Scott’s old serum [00:11:00] and hemoglobin, so the hemoglobin/haptoglobin band cut out from the gel and rerun to establish the polymers are stable polymers; they’re not something dynamic. You can see that each band, when cut out, runs again at the same place, quite a good gel, and quite a clear result Tuesday May 13th. [00:12:00]
On Tuesday, Wednesday, May 14th, I attempted an air-cooled vertical gel with forward and backward currents initially on obvious reason, so going five seconds forward, and one and a quarter seconds backwards, and then forwards, maybe to try to get mixing of some sort. But the results are really quite pretty, whatever I did, even though it was clearly a little odd, because it was on at 10 p.m., and by 11:20, the reverser had ceased to function, and it was constant forwards, so it was always going forwards at that point. But the patterns are good. [00:13:00] Thursday, Friday, May 16th, the VAC gel, vertical air-cooled gel, quite nice, not spectacular.
More and more examples, over the next few days. And sample on Wednesday, May 25th, labeled Emily [Atwood?] 667 plus hemoglobin, and showing that it’s type 2 [00:14:00] modified, or n sample. Again, a test of haptoglobin, whether it’s in equilibrium, or stable polymers on Thursday, May 22nd, and it says the result confirms that number one haptoglobin 2-2, and number two haptoglobin 2-2 bands do not revert to mixtures, so they’re stable polymers; the haptoglobin polymers are stable. [00:15:00] Again, thinking about this on Friday, May 23rd, query into conversion of haptoglobin types, so I mixed my serum, which is type 1, with Otto’s which is type 2, and type 2b, and compared with Dr. Scott’s, which is type 2a, and try to see whether they would come back to type 2-1 pattern, whether a 50/50 mixture of type 1 and type 2b would give 2a, but the result there is a possible indication, no sure haptoglobins. It was not, evidently, a very good experiment. [00:16:00] And so tried with semi-pure haptoglobins, and acid gels. This was the beginning of trying to understand the polymers, to see whether I could get something simpler. It’s actually quite an important gel. This was partially purified haptoglobin run in a gel which was 3 gels, pH 2.4, 2.1, and 1.7 acid gels, and the polymers present in the 0.02M HCl, negative, going the opposite way from usual, but the polymers are still present, and even at 0.05M HCl, although the gel is not so clear, it’s obvious that [17:00] the polymers are still present, and observable at an acid pH, so they’re stable; there is nothing that comes apart, if the haptoglobins are made acidic. On the other hand, and then similarly important — these are quite important gels for understanding what was going on in the whole situation with haptoglobin polymers, so that the Saturday one establishes that the polymers are not dissociable by changing them from negatively charged to positively charged.
And then the following day was a test on Saturday May 24th, by adding urea, and 2M urea, or 8M urea at an alkaline pH did not alter the polymers. So, of the type 1 — type 2a [18:00] and type 2b, they didn’t change to become 1, for example, so that they’re stable in the presence of 8M urea, and the comment is, “8M urea shows completely normal pattern, photogenic. Repeat with 4M,” it was not really necessary.
And then, here was an attempt to see whether higher ionic strength would make any difference, using [XSM?] buffer, which is much higher ionic strength than the usual borate gel, that it didn’t have any effect on the [00:19:00] haptoglobin types. The result is not very pretty, but it shows clearly that using XSM buffer didn’t make the samples dissociate; didn’t make the polymers dissociate. And nor did EDTA, in those days we called it “Versene,” it was a trade name for EDTA, and Versene (inaudible) sodium positives at that at pH 7, didn’t change the polymers, but trying 0.05M sodium hydroxide really did mess things up, and we couldn’t see what was happening in this, some very strange effects in that gel, the sodium hydroxide gel, [00:20:00] and — but EDTA itself at pH 7.5 did not take away the polymer, so they’re not held together by something that’s removable by a calcium or magnesium chelator. And then trying SS reduction, so I tried reducing conditions using thioacetic acid, SH-CH2-COOH, 0.1M, at a pH with boric acid. pH was 8.25 in the presence then of 10mM thiol. The polymers are still there, so they are not affected by this [00:21:00] simple reduction with a thiol. They’re too stable to be broken apart by just thiol reduction. Periodic acid oxidation was attempted with 0.5% HIO4, approximately 20mM, but this is the first (inaudible) comment is, there’s no detectable protein, but the gel was not kept, but it said, this is the first reagent experiment at a high pH that wrecks the haptoglobin, so periodic acid wrecks the haptoglobin.[00:22:00] And trying 0.1M HCl on Tuesday, May 27th, but [that option?] was bad; there’s no image there. So I think this must have been — it’s not clear, about 8-10 volts/cm of active paper, so this was probably a filter paper experiment of some type, absorption just as bad, because the following page shows some filter papers where haptoglobin was migrating 0.01M Versene at pH 7.8, or at pH 9.1, and just adsorbing to the filter paper, so not useful. [00:23:00]
Something about ripples of changes of voltage from reversing the voltage up and down, and testing anti-ripple current reverser, I was able to prove that the ripples are not due to heating. One pattern, plus 300 in one direction, put minus 300 in the other direction, gave no ripples with dilute hemoglobin, but if I changed the pattern, I could get ripples, inverting the gel to the natural position [00:24:00] gives a perfect result: no ripples, so that this was, the ripples are due to hemoglobin or whatever protein migrating upwards against gravity, concentrating, and then electro-decanting backwards, and so causing ripples. I can’t understand, it says here the result on — [some early?] book XIV when the vertical was [condemned?], anyway, test downwards migration more fully. And, this is obviously what was happening there, because on Thursday, Friday, May 30th, [00:25:00] the comment is, [two eights?] gel, two volt conditions downwards, and the image is May 20th, compared with the old, new vertical, versus the old vertical, and the results are virtually indistinguishable. Both are quite good. Looking at some, with two betas, the beta B and C, and the faster beta globin. [00:26:00] Friday May 30th, photography again. A rather pretty here, gel on Friday, May 30th, 31st, Saturday, vertically, air-cooled gel downwards with 135 volts over very satisfactory result, showing now three forms of the beta-globins, which I have looked previously, specifically commented in the notebooks, but there is a homozygote for slower, [00:27:00] and the heterozygote for slower, and the common one. Then there’s a homozygote common, and then there’s a heterozygote common, and the faster one.
So this is a pretty gel showing the first beta-globin, and the slower beta-globin on the same gel, and the homozygote of one of them, and the rather glorious resolution of the haptoglobin polymers, which one can count one, two, three, four, five, six, seven — these ten bands with common, very satisfactory. And this is thawed samples from previously. I must be getting close to the time when this was published, because I’m getting very good results, and I expect I’ll come across the gel that was used for publication [00:28:00] shortly.
On Friday, May 30th, I did an ultracentrifuge run on a large batch of haptoglobin 1-1, prepared in the usual way. Why, I’m not sure, it’s the results suggest about 15% heavy, and about 89% light impurities in the band. There are some impurities on the gel. And a sketch of the gel.
Vertical electrophoresis with and without the hemoglobin for the three standard haptoglobin done, fairly good, but [mustered?] just lateral, even, [of the slit?], exactly horizontally, [00:29:00] to whatever that means.
Purified haptoglobins on Monday, Tuesday, June 2nd. And [a good way met?] hemoglobin is written, it’s saying, quite difficult to understand at the moment, these gels. It’s 1% haptoglobin 1/1 from the ultracentrifuge, that’s what it is. And it is not very — nothing’s very pure. Purified haptoglobin.
Now this is really good haptoglobin, [00:30:00] purified gel, Tuesday, Wednesday, June 3rd and 4th, is a beautiful gel showing the four haptoglobin types with purified haptoglobin now, type 1, type 2a, type 2b, and type 2a modified. I know that this gel was published; I don’t remember where it was published, but that image is one that was definitely published — in fact, I think I do know where it was published. It was published in describing later with George Connell, the method of purifying the haptoglobin, because these purified haptoglobins were made by George Connell, and I will just check that.
A gel shown on Tuesday, Wednesday, June 3rd, 4th, is a very pretty gel, and was actually used in the publication of [00:31:00] by good George Connell, and myself on purification of human haptoglobin and quantitation. It’s, as the method says, the Dowex-2, X10, 200-400 mesh, and ionic exchange resin, positively charged resin, was used, as I’ve been able to show in the later books, was used as the chloride to absorb the haptoglobins from dialyzed serum at pH 4.2. And then the haptoglobin was eluted with 0.25M sodium chloride, after washing of course with water, and at room temperature. So these were beautifully-purified [00:32:00] haptoglobins made by George, and this particular gel was the one that appears in his paper, published in 1959 — we’re ’58, so published a year later, but it was a pretty gel that I was very proud of. Again, haptoglobin 2b showing one, two, three, four, five, six, seven, eight — more than 10 bands. I’m trying to understand how they were related as polymers, whether it was, you could find that the mobility was proportionate; the logarithm of the number, of the band or whatever, interesting. Very pretty [00:33:00] result. I remember being very delighted with it.
The ultracentrifuge run again on Friday, June 6th, with haptoglobin 2-2, good haptoglobin 2-2 purified, and rather pure haptoglobin 2-1, showing there were two main zones, not very informative, or useful. I suppose it was necessary, but it seems rather a waste.[00:34:00] On Tuesday Wednesday, June 10th, 11th, comparing the purified haptoglobins, rather dilute, with serum samples from which they were prepared. Some more gels comparing all of the samples that were done.
Sunday, Monday, June 16th, it’s rather nice to see who the samples are from. That’s myself, Dr. Scott, and Otto Hiller, prime examples of the three haptoglobin types, with and without hemoglobin. It’s a quite pretty gel; I don’t really know why I was running [00:35:00] it at that time, particularly because by then it was well-known, everything that that shows. Photography of one-dimensional gels, still being pursued, and more vertical gels. Probably thinking about getting ready for publication, quite a nice set of photographs on the Friday, Saturday June 21st, note the post-albumins, which are quite good. No particular family or anything.
I tried to electrophorese the zones out of the gel on Monday, June 23rd, 24th, [00:36:00] not too successful, so I froze and thawed the gels, which is the easy way to get something out, just freeze the gel, and thaw it, and centrifuge it, and you could get a supernatant which contained much of the protein that was in the gel, so freezing, thawing was adopted.
Rabbit-4 was run, rabbit serum, not worth a great deal of comment. But rabbit serum with hemoglobin and without hemoglobin. I never followed up. [00:37:00] It says, rabbit haptoglobin could be prepared in the same way as human, similar in purity; meaning that you could use it to make different species of haptoglobin. Haptoglobin has a rather high — a rather low isoelectric point. It’s still negatively charted at the pH 4.2, which is why it is absorbed to the resin of that pH, because it’s still negatively charged.
On the Monday, Tuesday June 30th was a gel, was a good image of a gel. And this is a whole set of [00:38:00] images which had been found at an earlier place in one of the other notebooks. It’s a stack of images that were just labeled “Gel 1, Gel 2, Gel 3,” and all loose together. But the actual gel itself on the record of it is on June 30th, July 1st, the same one. More attempts at getting good images of the vertical gel getting ready to publish, clearly thinking about it, and working to get good images.
And in fact on Thursday, Friday [00:39:00] July 25th is the gel that was finally published, as illustrating the vertical gel electrophoresis, it’s an interesting one, because, in fact, it contains samples from the Dionne quintuplets that were famous in Canada. In those days, before in vitro fertilization and many multiple births, quintuplets were extremely rare, and Dr. Norma Ford Walker had been studying these families of the Dionne quintuplets. They were so famous that the trains that passed nearby where they lived would stop to allow [00:40:00] passengers to get out and look at these babies. (laughter) So, they were really quite famous. And anyway, here are their samples. They [weren’t?] identical quintuplets, and there is a description in my paper of what they actually were, which I’ll try to find. It’s somewhere here, yeah. Comments that the — no, it’s not that one. Hold a moment. My notebook just lists them as their mother, [Eileen Hargreeves?], and Jennifer, Janice, and Joyce, three of the four quadruplets. They were not quintuplets; they were quadruplets. Even then it was quite rare, [00:41:00] very rare.
Hiller had a comment in the publication, which included this image. Let me backtrack. This image was always used for publishing the vertical gel electrophoresis. And that paper there is a comment on what the samples are. And samples 1 and 2 on the left-hand side of the gel are from a pair of female twins, aged 45 years old. One is unmarried, and the other a mother of four children, and the haptoglobin and beta-globin types of these individuals are the same, and you can see all the multiple bands of the haptoglobin, they look as if they were the same sample, but of course, they’re actually from identical twins. [00:42:00] And then samples three through five are from the three female quadruplets, aged approximately nine years old at this point. A sample from the remaining quadruplet was not available. The three quadruplets here concerned, it says in the paper, are known from their red blood cell type to be non-identical. The haptoglobin types of the first two differ. One is 2-2 and the other is 2-1; on the other hand, the post-albumins of these three children are indistinguishable, and they differ from the twins, etc., etc. So this was used to show that this gel shows that there are many inherited differences that one can see in these samples that have not been studied, and that I never had time to work on, but other people did at [00:43:00] some other times. So it says that the evidence is presented, which suggests that genetic factors are involved in the variations in the different individuals of the serum proteins which migrate immediately behind albumin, what I call “post-albumins.” There’s a nice gel, a nice publication from it, and a rather happy experience with these quadruplets. And Norma Ford Walker’s family.
A test with ceruloplasmin, on Friday/Saturday, August 8th/9th, and some other proteins. This is ceruloplasmin — [00:44:00] I don’t know from whom it was obtained, though I suspect it was probably via [David Pulich?], and maybe came from Kunkel, who prepared many of these metallic binding proteins. It might have come from Alec [Byrne?], or Henry Kunkel, the Rockefeller, they were very generous with material that they made, and were well recognized for their contributions. Particularly, Alec liked to work with ceruloplasmin, the copper-binding protein. And there is a comparison that it’s rather heavy, is ceruloplasmin, and whether or not it really lines up in the same position with anything in the gel, I don’t know, because I never proved that [00:45:00] any band in the serum was copper-binding. The gels, many exposure of this gel, of which there are ones that are rather darker, show things better.
Fraction from a Cohn fraction. I’m not quite sure what that is. Looks like a mixture of haptoglobins in channel 2, but it’s not; it’s [00:46:00] — there are two gels here, and I see. One is various Cohn fractions, and the other is, no it’s not clear. There are two gels, but only one diagram. They aren’t the same. Let me try that again.
Friday/Saturday, August 8th/9th, there are images of two gels attached to this part of the notebook. The comparison is of Otto Hiller’s serum with [00:47:00] ceruloplasmin types 1 and 2, ceruloplasmin 3, and Scheinberg’s ceruloplasmin, three different preparations of ceruloplasmin, the copper-binding protein that is absent in Wilson’s disease. I think these probably came from Henry Kunkel, but they might have come from Alec Byrne, who was an expert in Wilson’s disease. And the three ceruloplasmins are very, very similar, but one and two, and three are I, II in the text there, and three are slightly different, but they are obviously migrating in front of beta-globin, but whether any band in the serum pattern is the same, [00:48:00] can’t be established by just looking at things migrating in the same general place.
And then, there is a second gel there for which there are several images which are of purified Cohn fractions, with a comment that haptoglobin is in fraction IV-4, Cohn fractions, and so interesting but not terribly important.
Some saturation tests on hemoglobin/haptoglobin, on Thursday/Friday August 15th. They don’t look [00:49:00] very convincing. This is addition of pure haptoglobin 2-1 to hemoglobin, saturation tests, and in all of them, there is an excess of hemoglobin. But, stained also with benzidene, so there’s a benzidene test as well, but it’s not a very important result.
And the last entry in this book is again, typical of looking at ways of getting samples into gels, trying to introduce the sample laterally. [00:50:00] Semi-automatic insertion, doesn’t look very good, and there’s no image of any gel there, so that’s the end of Book XVII.