Oliver Smithies:[00:00:00] This is the beginning of Book 5, starting in January 1955. It takes up the [name redacted] family again, which I commented at the end of Book 4, didn’t appear to have a sketch, but here is more information about the [name redacted] family, and it starts off by saying, “The twins are again indistinguishable,” and comments on what the pattern looked like, and a little diagram on what the samples were on page 2, Tuesday January 11th. And “HP0” is written in in crayon on top of them. That means, later on we realized [00:01:00] that that these had no haptoglobin visible, and therefore we called them “HP0.” Newborn babies have a very low haptoglobin level, and so those nondetectable, and we used to score them eventually as “haptoglobin 0,” so the [name redacted] twins, [name redacted] and [name redacted] have been studied, and then Otto Hiller, of course, again, used often as a control. And the father and mother, [name redacted] family, and then Casey Kok, who was also a control. Otto, my recollection was what we later called “haptoglobin 2-2,” and Casey was “haptoglobin 2-1,” as I remember; it might have been the other way around. [00:02:00]
Some comments on the actual patterns themselves, nothing very critical. For some reason or another, I had an insulin experiment left over here, which I don’t remember what it was about. But it continues on page 4, and some little comments on it, not very critical.
Moving on, still looking at insulin, so I haven’t quite given up the idea of working with insulin yet. That is a decision which is going to come, but has not yet arrived. Looking at, on page 6, January 12th, sample of insulin on the usual 1mm [00:03:00] slot, 10V ascending a perfect setting-up for an activity test. I wanted to cut out the different bands that I got from insulin, and test them for their activity biologically.
Going on with electrophoresis on Thursday, January 13th, I’ve got insulin I think here. Can’t quite see what this is, “NAP–“ “no P.” No picric acid; I’ve got picric acid, amido black plus sodium picrate, and amido black. Why I was doing that was not at all clear, and it’s not very important. But I was evidently [00:04:00] trying to influence the migration by adding a dye, so the insulin migrated at a single band when there was sodium picrate there, and when it was amido black, and sodium picrate, it migrated as two bands, and when it was amido black alone, it migrated as a single band, slower. But evidently, I wasn’t happy with it, because I say “No good pH, plus picric acid decreases enough to alter amido black, [V?] set-up,” et cetera, et cetera, trying to understand the electrophoresis of insulin and its bearing on the function of the protein.
Going back to serum, [00:05:00] now beginning to be more concerned about serum proteins, Friday January the 14th, page 10, I got some more OS blood.
Now trying to, because I’ve seen that there is a red color deband when there is hemolysis in the sample, I’m beginning to learn about the interaction of the haptoglobin, that it turned out to be with hemoglobin. So here I’m talking about getting 12mL of blood, centrifuging, sucked off the [00:06:00] plasma, et cetera, washed the red cells with sodium chloride, and made a preparation of washed red cells, and I put them into the freezer to see to lyse the red cells. And I centrifuged the serum and the red blood cell, et cetera, and started to take serum and add different amounts of the red blood cells, presumably hemolyzed, to the sample. So here on page 12, I have increasing amounts of hemoglobin added to the serum, where I could see a faint single [00:07:00] band at the lower levels of hemoglobin, and as levels of hemoglobin are increased, I can see two bands, which are — now “no” means that one was hemoglobin-haptoglobin, and the other was free hemoglobin, but I don’t yet understand this completely at this stage, I think, except that I know that I’ve got a hemoglobin binding protein, and that it can be saturated at something, at a high enough level of hemoglobin.
Here we are back running samples on page 14, Monday January 17th, samples from Dr. McVicker, I think it was, “McV,” I’ve got, and George Beaton, Mike [Macamay?], these are various people [00:08:00] in the lab, and I’m beginning to explore the idea that there is a male and a female type. I guess I’ve got “Dr. VM,” and George Beaton, F1, and Mike Mac, F2. And I should retract that to exploring; I already at this point know that it’s not male and female, because F1 and F2 are what I used to call a female type, and it’s two males.
More gels on Tuesday, January 18th, George Beaton, MM, Mike McAllister, I’m running these samples again. [00:09:00] Still using these samples to try to improve the separations, or comparisons.
So here is January 19th, page 18, more experiments.
Continuing on January 20th, similar type of experiments, with a comment on page 20, January 20th, “740 being off, perfect as may be, GB still not as good as before. Note, this was seeded at 1-2,” talking about whether the [00:10:00] serum was obtained after clotting the blood with a seed of filter paper, which I used to have to do when I prepared the sample in tubes coated with fair petroleum gel, petrolatum, that is.
I was rather happy on page 22 to find a good sample here that “This revealed triple-alpha 1-1 in McV.” That was a notation that later on, I dropped; I didn’t know these samples — what the bands were, of course.[00:11:00] There’s a titration on page 22, was again a titration of the hemoglobin-haptoglobin situation, adding hemoglobin, to [McVickers’?] serum, and one can see the hemoglobin-haptoglobin band, and then the hemoglobin band itself.
Now then, [00:12:00] I ran, on this Saturday, January 22nd, a typical Saturday experiment, adding hemoglobin to one of the F1 samples, George Beaton, and one can see from the sketches that the hemoglobin-haptoglobin, as it turned out, complex, gave multiple bands, so I can see the pattern there. And I make a note, per note that, “This was the test with,” what I called, “an ‘F1’ pattern, ”which is the heterozygote, whereas the previous tests were an F2 sample, a homozygote — HP2-2, as we later realized. [00:13:00] And I was a bit surprised to see the pattern.
Monday, January 24th, still worrying about these, the patterns, and adding hemoglobin with a comment at the end, with a big circle around it, that “F2 is not equivalent to F1,” meaning that the pattern of the bands, the hemoglobin-haptoglobin bands [00:14:00] migrate at a different rate, a different extent in the F1 and the F2 sample. Simple F1, F2, and M test on January 25th, with something that looks like a sketch, but I don’t think it is. I think it’s just how the samples were inserted. The diagram is of the slots, not of [00:15:00] the pattern, of the resulting electrophoresis.
And again, some tests on page 30, with no sketches of the result, just comparing M, F1, and F2 on separate gel.
On page 32, I’m beginning to have a sketch again. It’s just commenting on the electrophoresis, beginning to worry about this staining fading. There was really no evidence of that. [00:16:00] Nothing particularly important, as we proceed.
Saturday, again, January 29th, 5mL samples from a family. Presumably now I’m working with Dr. Norma Ford Walker in The Hospital for Sick Children, but I don’t have any comment of that in this notebook at this point, just 5mL samples from Mr. and Mrs. [name redacted] and [name redacted], child aged 12, and twins, [name redacted] and [name redacted], aged 9; obviously not identical twins. Unfortunately, the [00:17:00] clotting was very bad, and bringing across in 10 degrees Fahrenheit were — so I evidently was getting this from Norma Ford Walker in The Hospital for Sick Children, and that comment, “Bringing it across in 10 degrees Fahrenheit weather, plus leaving until Monday (with sickness),” don’t know what that means. Maybe I had some fever or something, not conducive to good sample. Trace of hemolysis possible in the mother and father samples. But, not enough, my female advisors on color to see any pink, just darker in color. So I’m having this problem of getting the samples to clot, [00:18:00] so in the future, deliberately add filter paper before taking the sample, and clot at room temperature in order to get good serum. This is a constant worry of how to get serum without hemolysis. But it’s however, interesting page, Saturday the 29th, because it comments on going to the hospital, and the weather in Toronto at that time.
Actually, I was so concerned about the samples getting hemolyzed, that at one time, I was worried about even the slightest shaking, so I took the samples in the hospital with Norma Ford Walker, and then brought them by car, so as to avoid the temperature problem, [00:19:00] I brought them by car to Connaught lab, and I didn’t stop at a stop sign quite deliberately in order to avoid shaking. Quite silly, but that’s what I did, and I got picked up by a policeman, (laughter), and given a ticket for going through — not stopping at the stop sign of the highway, of the main road, and I think it was — I forgot the name of the avenue, it was a north-south avenue in Toronto, the biggest one. But I thought I could get away with it, so I went to court, and told the judge what I’d done, but he didn’t let me off; I paid the fine. [00:20:00]
Monday the 31st, running the samples that I collected. (laughter) With a comment, “very disappointing.” Looks like the father and mother, F1 are hemolyzed, and [name redacted] ditto, the twins, et cetera, et cetera. Looking at the plus or minus hemoglobin test, the best solution is to add hemoglobin deliberately to these samples.
Moving on, Tuesday February 1st, now with adding hemolyzed cells to the serum sample, [00:21:00] the family’s still under investigation.
Page 44, February 2nd, “Still worry about adding hemoglobin,” with diagrams of the result on page 46, with an excess of hemoglobin, 660mg/mL, is what I wrote down here. I don’t believe that; something funny about that number. But here, the patterns are [00:22:00] written down, with a comment that “all as expected.” Beginning to understand that the level of haptoglobin is not the same in all of them.
On page 45, the M’s, that’s the 1-1 haptoglobin, the M’s seems to show variation in the amount of hemoglobin binding protein amongst themselves, and with a comment, “as do F’s in the alpha-beta proteins,” still not completely clear on what’s happening, but there’s a variation in the amount of haptoglobin in different persons, is what I’m beginning to understand.[00:23:00] Evidently liking family with twins, because here’s Saturday February 5th, samples run again with a family with a twin. Some rather complicated comments.
Trying voltage gradient experiments again on page 54, measuring the voltage at different places at different times. [00:24:00]
And then on page 56, I begin to think it would be interesting to use Oil Red O to stain, to see if there are any lipoproteins. “Try Oil Red O in 70% pyridine, a good solvent, and also try osmic acid for fat.”
Trying a different buffer here on Wednesday, February 3rd, page 58, barbiturate buffer, with a rather different looking pattern, but not as different as one might think, but mainly the [00:25:00] difference is how far the albumin goes, and how extensive it is, trying to get rid of the splitting of albumin, not very successfully, because at 0.012M, the front of the albumin in sharp, and the back is very diffused. And at point 0.008M, the back is sharp, and the front is diffused, so that it’s obviously concentration-dependent.
And I’m thinking about bacteriophages for some reason. Let’s see if we can make it out. Bacteriophage preparation, see Thursday. Now I wanted to see if bacteriophage would enter the gel, I think. [00:26:00] This was, I don’t know where I got the haptoglobin, where I got the bacteriophage from, some little sketch of what was known of it.
Thursday, February 24, barbiturate gel, and put on a bacteriophage preparation on the gel with 0.008M barbiturate. Looks as if staining will be no problem. [00:27:00] Blue, but easily seen against white, et cetera, et cetera. My comment is that after running it off at one and a half hours at 6V/cm, the paper is stained, especially on the anodic side, but there are no bands. The bacteriophage didn’t enter the gel. Tried adding serum, I think as well, serum plus bacteriophage, it looks — that was done.
Page 62 is rather complicated. Reviewed and tested on March 3rd, 1955, different batches of starch. [00:28:00] Here I’m beginning, but I have not yet made comments on the batch numbers of starch, because I had evidently three batch numbers, three samples of starch, reviewed and tested March 3rd, 1955, one soluble starch according to Lintner, labeled so on so, lot number so and so. And, commenting that this sample is hydrolyzed to the extent, because Lintner method hydrolyzes the starch partially. This sample is hydrolyzed to the extent that 15%, the usual percentage. It doesn’t form a gel on cooling; it remains liquid.
And then another sample here, soluble [00:29:00] starch, according to [Lint?], “special for diastatic power determination,” et cetera. This sample is better than one gel this form on cooling, but the strength of the gel is poor; still too much hydrolysis. Sample 3, in reagent soluble starch, suitable for iodimetry. This sample is insufficiently hydrolyzed. It’s not practical to pour the product, obtained at 15% on heating, it sets into a semi-solid before cooling. So general comments, “Sample treated about midway between [two or three?] may well be satisfactory.” I’m beginning to realize that Lintner starches can be made of different gelling properties, depending on the degree of (inaudible), these three samples [00:30:00] were [Merck?] soluble starch, obtained so I was buying such that starch sample. It was a barbiturate gel, on with serum and with bacteriophage. A comment that, “17% gel, this was very fragile, ought to be tested instead of 15%.”
Saturday, March 5th, must have been with Norma Ford Walker, 5mL blood from the [name redacted] family, [name redacted] and two twins again, because Norma was interested in twin families, [name redacted] and [name redacted], 10 and a half [00:31:00] years old, using room temperature and filter paper technique to get good clot, good clot examples free from hemolysis, but I still have a comment that “clotting is still difficult; try glass beads next time.” And so I centrifuged these samples collected on Saturday, on Sunday, and “All three rather dark, but can’t tell if hemolysis is present. All the usual people cannot see any pink.” My color vision deficit is showing; I always have to ask if they’re pink.
Running the samples on Monday, and tested a BDH reagent, soluble [00:32:00] starch. They were weak. And, Merck, according to Lintner, an old batches he used by [George Connell and Gordon Licton?], would probably be OK at 16%, though not much stronger, so worrying about starch.
Running the family on Tuesday, March 8th.
Aha, beginning to think about getting photographs here, Thursday, March 10th, sample with Merck reagent soluble starch, trying to hydrolyze it with 7.5% HCl at room temperature, and [00:33:00] take out the sample at various times. That was the insufficiently-hydrolyzed starch, that I’m trying to increase the hydrolysis. (laughter) The methyl sera had partly dissolved by the morning, comment over, “Tested: too hydrolyzed now. Does gel, but a weak gel,” so I’m beginning to see that I can control the hydrolysis. To check this, I left some of the same source starch in distilled water overnight, dehydrated it with acetone, to find out if that affects the gelling, and it does not, so standing in water and re-drying doesn’t [00:34:00] affect the gelling. So watering is OK.
More starch tests, page 72. And 74, with a good diagram, and not quite as usual; can’t predict what to alter, though 16% at 0.04M borate would be a guess, increase the starch to close the gap between alpha-2 and albumin, and increase the borate to compensate and de-sharpen the back of it, so I’m realizing that you can vary the starch concentration, and you can vary the borate concentration to [00:35:00] influence the sharpness of the bands. Later on, when Otto Hiller began to make the hydrolyzed starch when I moved to Wisconsin, we would describe what concentration of borate, and what concentrate of starch to use to get a standard pattern. It would vary a little bit. That was always around about 0.03M borate, thereabouts. I wanted to try to clear the gel, make it transparent, so I tested urea, but the dye washed out, and in any case, the clearing was poor. Trying to make the gels transparent, [00:36:00] but after staining, urea didn’t help because it removed the staining too.
Comment on page 76 of mice and age groups, and families, and other Saturday experiment, again with Norma Ford Walker’s family, and another pair of twins, identical? And then everybody was M, simple pattern. Father, mother, and the twins who were about 11 years old.
Tuesday, March 16th, did some tests with a spare gel.[00:37:00] Monday April 4th, well first of all, Saturday, April 2nd, blood from [name redacted] family, fraternal twins, aged 20 and 21 years old. No visible hemolysis, set them upon Monday, and type them.
I haven’t made a comment on this, but my electrophoresis system was still rather complicated, that the electrodes were silver/silver chloride electrodes, in a rather strong salt, with a bridge to solution of [00:38:00] the borate, and then a filter paper bridge to the gel samples. And really, the use of silver/silver chloride electrodes to avoid a pH change was not necessary, but I didn’t know that, and still making rather complicated silver/silver chloride electrodes, and making four new ones on Thursday, April 7th. A new insertion idea, tested roughly, promising, but I tried to use agar to help insert the sample; wasn’t very happy with it. [00:39:00]
I’m thinking of different buffers still, looking at proteins now, Tuesday March 3rd, lactoglobulin, but I don’t really know this, 0.0015M potassium hydrogen phthalate probably, just a slight (inaudible) zero. Insulin, in sodium chloride, so lactoglobulin in potassium hydrogen phthalate. [00:40:00] And made a sodium chloride gel. Comment, “use sensible buffer, at least for the test,” so I made a sodium phosphate, NaH2PO4, and Na2HPO4, pH 7.12, tried different amounts of buffer, and comments on working at what pH.
But, I have, on page 86, I have available 0.02M in phosphate buffer at pH 7.3, use this at 1000M, and made a gel, [00:41:00] a 13% gel which had a pH of 6.9, but it was worth trying, because 6.931 on April 1st, was nearly correct, and was unbuffered. I had a 13% gel, pH 6.94, and with a comment, “This is unexpected; must be there’s some phosphate buffer in the starch anyway, can use 0.002 –“ unimportant comments now, that worry me at the time. [00:42:00]
On Wednesday, May 4th, sodium phosphate gel, different pHs. Looking at the migration of insulin still. On page 89, comment on the insulins, “At least the osmotic gel, is visible at about 3.9cm, 330, so look photograph just to see, proves not to be the insulin.” I was looking at the gel and [00:43:00] seeing that I could see some shrinkage of the gel; that’s what I mean by (inaudible) osmotic zone, but it turned out not to be where the insulin was; it was just a front of some sort. But page 89, “The resolution is better after four and a third hours, but there are some little things that are worrying me, absorption and desorption, it’s the pH changes that (inaudible) later (inaudible).” [00:44:00] I tried doubling the phosphate concentration, since the current is still within acceptable limits.
These results are shown on page 94. I’m still getting a variation of a single band, or more complicated patterns from insulin, and 93 commenting that, “The results suggest that this true resolution is only when you’re near to the isoelectric [00:45:00] point where the solubility is low; therefore try going to the acid side of the isoelectric point, or add something to the gel to increase the solubility; for example, ethanol.” I don’t know why I thought that ethanol would increase the solubility.
Tried some of Dr. Scott’s extract on page 96. Friday, May 6th, worrying about solubility, and Dr. Scott suggests that 10% glycerol will keep the insulin in solution, so I made a 13% gel with 10% glycerol, sodium [00:46:00] phosphate buffer. It sets rather sticky, but not sticky when properly cooled. Also made a gel with 20% glycerol, but it was too viscous to make a good gel.
(inaudible) was that, or quite commonly that my pages are often run consecutively in reverse, as it were. So page 98, on Friday, March 6th, is the beginning of those experiments. It continues on page 97, so 97 is the result of all the sequel to what happened on page 98, [00:47:00] some osmotic problems. But the gel, and the paper was stained largely still insoluble. The insulin just didn’t migrate at that pH, insoluble. Tried some mouse blood. Bled from the jugular, with survival.
More insulin tests on page 99, getting these families rather regularly now with the help of Norma Ford Walker, here again Monday, May 9th for the new family. [00:48:00] This is [name redacted] family, father and mother, and son and daughter, older children, 24 and 19. And the mouse serum plus hemoglobin. Not happy with the results, and repeated everything on Tuesday, May 10th. This is experiment on again with all new solutions. Mouse [00:49:00] samples taken on Tuesday, May 10th, with a note, “See May 12th.” I have more mouse samples. I don’t at this point remember having done the mouse tests.
Wednesday May 11th, setup using new starch, checking the F1/F2/M classification of the samples, father and mother [name redacted] family, with now a different one, keep [name redacted] as [00:50:00] standard F2, and [name redacted] as standard F1.
Keep Thursday May 12, bleeding more mice. Try some other strains of mice, at sick children’s hospital, Banting, Best et cetera, et cetera, but there are no results that I can see anymore; they’re just samples, but no sketches of the result.
Serum from rabbits, [00:51:00] rather haphazardly doing insulin, and mice, and rabbits, et cetera, on page 110, set up an insulin experiment and bled six more mice.
With the insulin, I have a comment here on Friday, May 13th, that since 31% ethylene glycol showed the greatest solubility for the insulin, I decided to [00:52:00] make a last try at a greater concentration, so I made a 40% ethylene glycol phosphate buffer gel, gelled very slowly, very weakly, and not worth trying 50%, as it will probably not gel.
Some sketches of the hemoglobin, the red bands in the mouse samples on page 111, clearly plus hemoglobin, [00:53:00] with a comment that all the mice are approximately this equivalent, considering difference in hemoglobin amount. So we’re getting another strain; the blood must have settled.
More samples on Monday, May 16th, from another family, this [name redacted] family, hemoglobin pattern. And with a little sketch on 113 shows nicely the nonequivalence of the hemoglobin-haptoglobin bands in F1 and F2. [00:54:00]
On page 116, the insulin result, not clear what they are. It’s whether adding insulin to a serum and dialyzing it makes anything bind; I think Dr. Scott was very interested in a serum, the possibility that blood contained an insulin-binding protein. I suspect that’s what this experiment was about, although the documentation is not good enough for me to [00:55:00] determine that at this late date.
On Tuesday, May 16th, trying to see what the results are. Insulin dialyzed against distilled water, with serum, or without serum, or with horse serum, and rabbit serum alone, rather heterogeneous experiment. [00:56:00] A little family, confirming the family typing on page 118.
It’s rather interesting in looking at these now, I don’t remember these experiments at all well, because they really never led anywhere, so they didn’t form much of a lasting memory in my mind.
Still looking for my precursor for insulin, [00:57:00] Tuesday, May 24th I’m starting to homogenize pancreas, 4g of frozen, minced pancreas homogenized in 25mLs of 0.9% sodium chloride, et cetera. And what we expect to get, is this should give 100% convulsions of the test mice at 1-10 dilution when activated. And here are the tests, the standard on page 123, reading these. There is a fraction there. That’s the number of convulsions per number of animals, so the standard on page 123, so the standard solution of insulin [00:58:00] diluted 1/20 gave 15 out of 24 convulsion mice that fell out of the assay cylinder. And I think I’ve described the assay cylinder before, but in case I haven’t, the assays were carried out with rotating cylinders, probably about two feet along with a wire mesh inside, and rotating. Mice were put into these cylinders, and when they were normal, and unaffected by anything, they could easily maintain — not fall out of the cylinders, because they could run as the cylinders rotated, and could catch hold of the wire mesh. But if they had [00:59:00] insulin in excess, they would become convulsing from hypoglycemia, and they would fall out of the rotating cylinder, when they’d immediately be caught by one of the technicians, and given a shot of glucose to make sure that they didn’t die. So the mice were used to assay insulin; this was the only good in vivo assay of insulin available at that time. So on page 123, I see the standard 1/20, 15 of the mice out of 24 fell out of the cylinder, and that corresponds to 18.7 units of insulin from standardization curve. And my homogenate [01:00:00] alone, 2 out 12 fell out of the cylinder, corresponding to an insulin level of 5.0 units. And when the homogenate with serum, 3 out of 12 fell out, but one died. So that means that the actual assay was then the same as 2 out of 12, and 5.0 units. And retested at 3PM, and got, instead of 15 out of 24 for the standard, got 19 out of 24, homogenate alone 5 out of 12, and with serum 1 out of 12. And, [01:01:00] the acid alcohol extract homogenate 6 out of 12. So I’m trying to make insulin, and looking for the precursor.
But also, quickly transferring my attention to serum again. So on Wednesday, May 25th, I’m setting up BALC mice with and without hemoglobin, female. Bled some more mice; the results aren’t really shown.
Thursday, May 26th, [01:02:00] bottom of 126, page, shows what was set up, with an acid gel, pH 3.85, a formic acid-buffered gel, and on the next page, what I observed.
On page 127, with the comment of this “as useless.”[01:03:00] Friday, May 27th, I ran a sample of insulin, and a sample of Richardson diabetic serum. And the results are sketched on page 132. The insulin results are not much different from previous experiments, [01:04:00] and these were samples of insulin mixed with serum, I think, a little bit obscure, or mixed with lysing extracts. Homogenization I think, not very clear. Also, not very important.
More families, May 30th, Monday usual after having got the samples on Saturday. [name redacted], 14 year old son of the last family, blood taken Saturday and centrifuged Sunday, et cetera, and also samples from the [name redacted] family, [01:05:00] with BALC mouse, comments that —
Looking for differences in the hemoglobin binding pattern of mouse serum on 133 confirmed that the BALC as an equivalent to the [KNO?] mice, and the hemoglobin binding is clearly defined as stronger in #6, which is — (laughter) I don’t know what #6 is, then #7. 6 and 7 are different mice, I imagine. [01:06:00]
Trying acetic acid-sodium acetate buffer, pH 4, with insulin again. Still trying to get better electrophoresis with insulin.
Wednesday June 1st, lysine in acetate buffers. pH is 3.97, 4.13, et cetera. Possibly diphasic band, but it may be an illusion anyway, clearly no significant in view of the others’ patterns, so not doing very well with insulin.
Trying again Thursday June 2nd, [01:07:00] 4.43, 4.57 pHs with lysine or without. And, a comment that “I suspect that the only effect of lysine is on the conductivity of the gel, doesn’t really alter the pattern.” Thursday June 2nd, at least there are decent sketches of what the results are. “Therefore, put the pH up by two further increments and see what happens.”
I don’t know why I’m particularly interested in the diabetic sample, but I am, Friday June 3rd, test of [JU?] starch and [name redacted]’s diabetic serum. This is, the fact that [name redacted] is diabetic, [01:08:00] and his name appears is part of the reason that the scanning of these books was interrupted in the first attempts to scan them at the University of North Carolina library, because they felt that having the name of a diabetic person without permission from that person would be unethical, even though these are more than 50-year-old records. That subsequently was decided to be not a sensible means of deciding whether to scan the journal, and so, as we are doing now, the books have been scanned completely by the library. And I’m recording this on [01:09:00] July 14th, 2015. All books have now been scanned by the library.
Going back to the book itself, Friday June 3rd, just comments on how the electrophoresis worked, but no sketches.
Saturday June 4th, 2mL of blood from infants yesterday. [name redacted] 14 months, and [name redacted] 8 months. They have haptoglobin all right, because I’m able to type them as F2 and F1. So by that age, 8 months and 14 months, they have haptoglobin, as I would now [01:10:00] call it.[name redacted] family, so — it says, Saturday June 4th, Monday June 6th, different families, [name redacted] family collected on June 4th. Nothing very remarkable. Tuesday, June 7th, [name redacted] family, so I’m getting lots of families. The [name redacted] are all the same, maybe F1 with first beta query due to high temperature, couldn’t get a good match with past sample, but most likely they’re F2 throughout, so it’s clear that [01:11:00] there’s been some confusion about the labeling, because on page 148, one can see some fainter label by pencil has been overwritten, so F2, F2, F2, looks like everybody is F2, except in the [name redacted] family, and [name redacted] is F2.
Another family, June the 8th, nothing very remarkable.
June 10th, another family. Lots of families; lots of work. It’s under here “might be hypothyroidic,” and a Japanese family.[01:12:00] And very clear results, no sketches, but just the typing on Monday, June 13th, of these families, of the [name redacted] family, and [name redacted], Mrs. [name redacted].
And on Tuesday, June 14th, the Japanese family.
Page 159 and page 158, typical results [01:13:00] comparing different samples, just to be quite sure that they are as typed, comparing them against samples that have been picked as being standard F1 or F2, or M, and checking them, three samples per gel. Nothing very remarkable in what’s happening here.
Page 160, rough test on the new batch of BDH, British Drug Houses, starch #12294. [01:14:00] I thought it was rather under-hydrolyzed, and set solid.
Evidently training somebody, because I’ve got a comment on Tuesday, April 26th, page 166, “Let Gerry practice on this tomorrow, and set up the equivalent three flasks.” I don’t remember who Gerry was; it may turn up in a little while. These were some people with problems, because here’s one person with scleroderma, and a person with rheumatoid disease, and a person with acute ACDIS, [01:15:00] lupus erythematosus; I can’t translate that as I should be able to, but I can’t. “ACDIS, lup erys,” and hemoglobin, no particular comment on the result.
Beginning to hydrolyze starch again, to make my own, on page 167. This was a 38, 39, 38.5 (inaudible) degree, 330g of starch, with 600mL of 1% HCl and acetone; I think that’s what [01:16:00] it is; it’s not very clear.
See if we can find out better what it is on page 168, Monday, April 25th, 200g lot of starch, 16, 15. This is testing hydrolysis, testing the starches, all three made readily, “best yet,” and approximately equivalent, usual. So these are the hydrolyzed starch, made previously. Page 167 shows what I’m trying to do with this, as 330g of starch per 1000mL of 1% HCl in acetone. [01:17:00] The total volume is 1000mL, but it was 330g of starch, and 600mL of 1% HCl in acetone, and stopped each by adding 1M sodium acetate that had been filtered through a filter, #51 on paper.
Testing the new starch on Friday, April 22nd.
Oddly enough, I see the problem here. [01:18:00] Aha, this is why this had been difficult, because this is — I’ll go back and start again on this, because it’s the problem of starting at the back of the book with one set of experiments, and from the front of the book with another set of experiments. So let’s reorient this book. The experiments up to June 15th are straightforward experiments in sequence, ending on page 161. That is June 15th of the year. But page 162 [01:19:00] is April 28th, so it’s coming in the reverse direction from the starch, so I’ll resume that in a moment. I’ll just take a little break.[00:00:00] So, this is Book 5 again, but starting at the back of Book 5. Here, the experiments were related to starch preparation. Begins on page 215, that’s the beginning, and we’re going to be going backwards from there. “Cellular starch, used to date is probably potato starch. Not equivalent microscopically to corn starch, but equivalent to potato.” So I was trying to determine what was soluble starch was, versus starch which I knew was from potato. But I tested Fisher corn starch at [00:01:00] 5%, 7.5%, doesn’t gel at 5; gels weakly, but is already almost too viscous to handle when unhydrolyzed; that’s Fisher corn starch. And BDH potato starch in water; very viscous 5% OMS gel, but very sticky and useless, 7.5% too viscous to handle, showing that unhydrolyzed starch is quite useless of making gels. In certain, we probably come across it later, but in case we don’t — Jim Angle, in New York, and we certainly will talk about him later, found out that there were [00:02:00] machines available that would test the viscosity of starch as it was heated up in water, or in other solution, and could be used to determine the properties of hydrolyzed starch, and the increase in viscosity which occurs when you boil starch, hydrolyze starch, that is, is quite typical. So it goes through a phase of initially just a suspension of starch grains in the buffer, then as it heats up, it begins to gel. It begins to go into solution and make a very stiff gel, and as the temperature rises and gets towards boiling point, then the viscosity falls, and you have a homogeneous [00:03:00] solution that is viscous, but quite handle-able, and then that can be poured, and that will set into gels. And the gels can be either too weak, that’s obvious, or nice and firm, and break cleanly, which is ideal, or they might break in a sort of sticky way, which means they’re under-hydrolyzed. So over-hydrolysis would lead to a gel that couldn’t set properly, or was very weak; under-hydrolysis leads to a very sticky gel that can’t be sliced easily. Anyway, that’s what I’m trying to understand, starting on page 215 of Book 5.
So, knowing that Lintner starch is made by acid hydrolysis, [00:04:00] I set up 8 flasks of potato starch in 7.5% HCl for daily tests using et cetera, two volumes per volume of dry starch, mixed thoroughly initially, but don’t mix again, because it’d be difficult with large volumes, so just allow it to mix once and then settle. So, that was Monday, March 14th at 5:30, and then 12PM, Tuesday March 15th, the first flask was washed, and acetone-dried. So that was what I called “19-hour starch,” — this is page 214 — a 10% solution in water was readily made, and gelled, but was very weak, and tried 15%, result was a gel close to the [00:05:00] usual. Perhaps a bit weak, so 19-hour starch was a good beginning. And I tested under electrophoresis with [name redacted] serum, and I was pleased at the low count in the gel, but comment that, “the gel was definitely weaker than usual, but still can be handled.” This was 15%, approximately equivalent to 12.5% of a usual gel. So try 17.5%, and reset of a flask, the rest is potato starch at 12 degrees Centigrade at 4:30PM for cold hydrolysis. [00:06:00]
They’re going back to the, try 17.5%. On page 213, I say that 17.5% was made, and the results were, as usual, very pleased with this, perhaps 17% will be better. But anyway, needn’t worry; if I can’t get it the other OK. So I obviously feel confident that I can make my own hydrolyzed starch starting with raw potato starch, probably 12 hours hydrolysis at room temperature will be good.
And I tested the 10 degree hydrolysis, going backwards then, page 212; we’re on Friday March 18th, BDH sample, set up to 21.5 degrees [00:07:00] Centigrade for six and nine-hour hydrolysis tests. And, some 12 and 15-hour tests as well. “Tried tapioca starch, 10%, set solid and very glue-y, like tapioca pudding,” comment. I’ve got a comment here, “CS Hudson starch acetone, 1% HCl by volume; boiled for 10 minutes, it doesn’t gel. Methylcellulose, no good. Won’t go into [00:08:00] solution. And cellulose gum, difficult to dissolve,” trying different things, so trying tapioca starch and whatever CS Hudson is, and methylcellulose, and cellulose gum.
Next page, 211, setting up the tests of the hydrolyzed starch that I made. So, concluding there that at the bottom of page 211, “So the 15-hour hydrolysis starch is pretty close. Test with electrophoresis.” [00:09:00] Guess they were just gel tests. Continue with that.
Monday, March 21st, various gels being tested. Interestingly enough, notice that as you add starch to the HCl, it gets warmer, so heat is evolved, and therefore, in large lots, the starch to the HCl, to avoid the effect of heat rather than adding HCl solution to the starch. [00:10:00] Just the order of addition. Here’s the “CP” again, Baker’s starch CP, potato powder starch for iodimetry.
On page 218, I’d come to the conclusion that it’s not worth bothering with the cold tests. “Abandon the cold tests.” Three-day cold hydrolysis sample, a rough test, it’s set solid. And then, I like the starch from Baker, so, “Order all the rest of lot 8072 Baker, [00:11:00] and check Montreal for more [F-wash?] sample,” OK, I was trying to get the starch that had been used — that, it was difficult sometimes to trace the origin of the potato starches, and so here I clearly wanted to get all I can of one particular batch, and to see if I can get something from Montreal that was involved in the supply chain; I don’t quite remember how.
Tuesday, March 22nd, page 217, typical experiment. Merck starch, OS 15 hours, and 10 minutes starch and Baker-washed starch. [00:12:00] And the 15-hour starch is definitely a little too viscous for easily handling, and took them off, and had decided that Baker is no good.
Page 216, temperature for the latest hydrolysis was 21.5, and I have some 15-hour, 16-hour, and 17-hour starches, hydrolysis time, that is. And I got the rest of some other batch, 541117, set up with HCl, temperature increased to 25 on the addition of the HCl, [00:13:00] mixed well and left.
Wednesday, March 23rd, still testing different lots of starch.
Thursday, March 24th, two gels with BDH 12 hours, with known samples. And the results approximately indistinguishable from the Merck starch. Note that this, et cetera, is not so good, sample insertion cutter.
Friday March 25th, BDH-2, 17 hours, insufficiently hydrolyzed; 19 hours BDH-1, usual. [00:14:00]
Saturday March 26th, mixed up in the comment on getting a blood sample, from the [name redacted] family, non-identical twins, age 10, [name redacted] and [name redacted]. So with a little bit of doing samples, and running, testing starch and running samples, [00:15:00] mixed up here, so 210, page, Sunday March 27th, prepared yesterday serum, and there’s the father and mother, [name redacted] and [name redacted]. Testing different starches at the same time. And, repeats just to confirm them on page 209. March 29th, page 209, 208, insulin tests.
Still continuing to try some insulin tests [00:16:00] with the BDH starch, 17-hour hydrolyzed, on page 206.
Insulin tests again, Thursday, March 30th. Lactoglobulin also being tested. [00:17:00]
Friday, April 1st, continuing with lactoglobulin and insulin. Some sketches of the results on page 200, with a comment then, on page 199, to test 13% BDH-2, hydrolyze 17 hours with lactoglobulin and insulin, using two different gels, insulin to be in 0.0015M [00:18:00] borate gel with 0.001M sodium chloride, and the lactoglobulin to be 0.0015M, potassium hydrogen phthalate with two different pHs.
Trying 37 for the hydrolysis on Monday, April 4th, four-hour sample at 15% gels, quite quickly from a very watery solution, but the gel was weak. “Leave for overnight setting and test tomorrow.”
Looking at them on Wednesday, April 6th, looks OK. Three hour sample, “Two hour sample looks OK. Three hour too weak. [00:19:00] Two hour sample 15% gel on [name redacted] serum.” No comment on the result.
Until next page, Thursday, [name redacted] serum on, turned out to be what was later called “2-A,” that would be F1. Testing on 15% BDH two starch, one and a half hours at 37 degrees sample. [00:20:00] Begin to understand that there are differences in rupturing of the gel, of the starch grains, as I called it, because on page 195, Friday, April 8th, I made 12.5%, and 14% one hour hydrolysis gels, this is 37 degrees hydrolysis. Surprised to find that it’s not the gelling strength which is great of the starch, but that the grains are difficult to rupture. 12.5% gels very weak; not kept; 14% not very good. So, messing around with the different hydrolysis procedures.
Continuing still, [00:21:00] April 11th, 72 hour cold hydrolysis, retested, probably used for the 72-hour cold hydrolysis with two check marks and underlining, indicating it wasn’t useful; did not set solid. I must try the Hudson method, boiling acetone, plus 1% HCl, using sodium acetate to stop the reaction. And I tried this, but bumpy was very serious, therefore [00:22:00] try this acetone method at 37 degrees, instead of 57 degrees. Then took a batch in 100mL of acetone plus 1mL of concentrated HCl, 10 minutes at 38 degrees, added sodium acetate, washed it. And then on page 192, 10 minutes 37 degrees acetone starch gels perfectly. A bit too solid.
And then it says, on page 191, the results were almost perfect, as regards the albumin spreading the front of tray sharp, but granular again; the starch is a bit granular; therefore test room temperature thoroughly [00:23:00] as the hydrolysis is so fast, I can try cold also, if necessary. So I test 60 minutes at 25 degrees, too solid at 15%.
More tests on Wednesday. April 13th. Results on page 189, grainy, slightly grainy. All three were poor. The 37-degree hydrolysis.
Continuing this problem of finding the best conditions, page 188, 187, April 14th, [00:24:00] cold starch 10 degrees, 18 hours hydrolysis. Results very promising. Looking at the graininess of the resulting gel.
Friday, April 15th, took out number two cold, which is 41 hours 10 minutes, looks extremely promising on the test tube trial, but these were cold hydrolysis in the comment on the bottom of page 183 is that on examining the more closely, grainy all of them, but very slight grainy — the graininess of the gel. [00:25:00]
Some 37 degree test going on on April 18th, Monday. On page 179, I underlined that the results are promising; suggest that a carefully-controlled 35 minute sample would be OK. That’s 37-degree sample. Still continuing, April 17th, April 19th, [00:26:00] April 20th.
Here I have a comment that, 45-minute on page 173, “The 45-minute sample is the best yet. The only one image, post-alpha and first alpha-beta are resolved, and probably stronger than usual.” So, struggling, but getting very close to understanding how to hydrolyze the starch. [00:27:00]
Hydrolyzing, and then the acetone drying is a little bit of a problem, as indicated on page 171 that I evidently run out of acetone, because I had 200g starch with about 600mL of 1% HCl in acetone for about a trial decided as the acetone is still lacking; meaning I presume that I didn’t have enough to leave this steeping in distilled water, after setting up [00:28:00] for larger bulk, setting up in water is likely to be a good practice, but still not the end of these tests.
Still at it, Monday April 25th, a new lot, test at 16, 15, and 14%, 0.03M borate at pH 9.03, “All three made up easily, best yet, and approximately equivalent to normal.” pH of the 15% was 8.54, so this is [00:29:00] a new lot of starch, was made a little earlier.
37 degree one again, tried on page 167. And tested on page 166, but a new batch of hydrolyzed starch, BDH starch 12294. Here is where I already had commented on this, page going in the other direction, but [00:30:00] page 166, I tested some sample from scleroderma and rheumatoid disease? And acute dystrophic lupus erythematosus, I think [name redacted], mixed up with the starch tests, using BDH starch 12294, which I think was rather under-hydrolyzed, quite a lot under-hydrolyzed, set solid.
More tests of lot [00:31:00] #12294, trying to hydrolyze it a little bit more, I think. Not very clear. Yeah, 60-minute and 90-minute hydrolysis at 38.5 degrees, and still, at the 60-minute still much too little hydrolysis, and set solid, and at 90-minutes, still too little hydrolysis, set solid. Tested the diabetic serum again. [00:32:00]
And that’s the end of the backwards reading of this book, April 28th, page 163 is the end of the backwards portion, and page 161 is the end of the forward part of that book, so altogether, that’s the end of Book 5.