Oliver Smithies:
Book Three ended with still trying to get a good way of inserting the sample into the gel. And Book Four, 1954, continues with that problem. Although, for a reason which I don’t quite understand, the first sample started on January the 10th. No, we’ve got something wrong here because Book Three is ending Thursday [00:01:00] September 9. Book Four is beginning January 10th. All right, let’s start again for this. So physical Book Four starts off on page four and you can — continuation of September 10th entry in Book Three. The preceding pages, one, two, and three — one and two — are really later and I’ll come back to them at a later time.
So, here we are, Monday, September 13th in Book — physical Book Four. Again [00:02:00] just trying to find out the best way of applying the sample to the gel. Looking at the effects, also, of voltage gradient — so the experimenter on Monday, September 13 is to try a different voltage gradient. Gel is at 8.46 and six volts per centimeter, or 7.5 volts per centimeter — both OK, it says. Images are drawn by hand, as usual — show the difference in migration, but not really any significant difference in what’s observed in these [00:03:00] two cases.
Tuesday, September 14th, two tests made to determine if electrodecantation is important, and to find out the effects of a small load — that’s six volts per centimeter. I’m not sure what that exponent is about. The result is a sketch saying “not kept and not conclusive for electrodecantation, repeat this.” [00:04:00] With a summary of the status at this position, being on page — this is page 14, put in by hand at some later time — that the spaces have definite advantages over filter paper, but probably must be kept narrow to avoid electrodecantation. The six volt per centimeter results, with a 0.8 millimeter spacer, are the best to date. However, the practical difficulties with spaces are considerable. Cannot yet exute — fibrinogen is showing, so I’m still concerned about whether I can see fibrinogen or not. On Thursday, September 16th [00:05:00] on the line heavily six volt per centimeter plasma, with 0.25 millimeter spacer and the comment, “No clear fibrinogen.” I tried a slit with starch grained and I tried a slit with glass beads, and not very much difference between them that I could see at that point. [00:06:00] They tried fractionating the glass beads in 50% glycerol by mixing them and allowing them to settle. The product could be pipetted when wet, like the starch. If it’s better than starch, fractionate it more.
So Friday, September 17th [they?] test them, soluble starch supported material in the slot in one place and glass in the other, with a comment that neither looks very good and they interfere with each other [00:07:00] so try something else. Try untreated starch and perspect beads. So as we go on, September 20th, I’m trying two gels set up with potato starch and [lucite?] beads. And that’s not hydrolyzed starch, that’s just simple potato starch from BDH, or British Drug Houses. Later on, I had a lot of difficulties getting the right starch and I used to get unhydrolyzed starch from British Drug Houses. Anyway, here’s Monday September 20th with Lucite beads and potato starch using serum. And a comment with [00:08:00] “not kept but no signs of adsorption on these two. I can’t see any difference between them.”
5:40 PM on Monday the 20th, another gel set-up. A very careful test of gas beads and plasma. Six volts per centimeter, with a comment that, “It looks as if Lucite is really better than glass beads. Better check tomorrow.” [00:09:00] And there was a comment that it confirms yesterday, glass and Lucite, nothing kept. Looks as if Lucite might be a little bit better than glass, with a comic, “Lucite query, OK. Glass definitely odd.” Still worrying about it… Thursday, September 23, “Accidentally used coarse [00:10:00] soluble starch here. Did not discover until 5:00 PM.” I doubt it would’ve made any difference, anyway, but we’ll see. With a comment on that experiment that was done, on Thursday, September 23, “Quite hopeless. Might’ve guessed when such large quantities had to be run.” It was trying a very wide slot, or something of the order of five millimeter — a slot of five millimeters with serum or plasma. It’s much too large unless it’s dialyzed so that the gel was [00:11:00] badly overloaded. Meanwhile (laughs) the electrodes can do with improvement.
Much precipitate on the plasma electrode, presumably colloidal silver borate, and where that’s coming into it. New batch of starch, not kept gel, measure in voltage gradients again. And another conclusion about this whole business, “Even starch shows erratic alphas. Never try cut-out and narrow slits with no spaces.” Trying to get away from these problems of [00:12:00] inserting the sample. Monday, the 27th of September showed an attempt to measure voltage gradients using a platinum grid on a dynamic potential — potentiometer instead of the copper electrode. So it’s copper, copper borate versus saturated potassium chloride calomel with a platinum grid. [00:13:00] Unusual changes in voltage gradient where the albumin is so not due to the problems with copper borate, that I’m afraid about.
Typical experiment again of a paper, number three paper, of a slit and a slot with coarse potato starch as a filling material. This is on Tuesday, September 28th. [00:14:00] Prepared centrifuge serum from this tomorrow, and run three gels then. So Wednesday, September 20th has a gel with number three paper slot, a gel with a slit and a gel with coarse potato starch. The conclusions are that there’s — starch gives approximately the same results as a slit, so that it — starch grains are pretty well as good as nothing in the slot, which is what I eventually published, that starch grades were perfectly satisfactory — a little bit laborious, but satisfactory.
The three diagrams with the paper set in the starch are very, very [00:15:00] similar. I am beginning to understand that there is a binding protein because on October 1st, I do a sample — I do a test with hemolyzed serum and plain serum, and the gel sketches show that — a band that was definitely hemoglobin [00:16:00] combined with something. “Alpha-two plus hemoglobin” is what I call it, is in a different place than the alpha-two — what was alpha-one. So (laughs) notation is difficult. Alpha-one definitely decreases in the hemoglobin — in the presence of hemoglobin, and I call the other alpha-two plus hemoglobin, but it’s the same protein moving from a slower position to when combined with hemoglobin, moving faster because of the hemoglobin charge. Even though as I now know, the molecule is bigger.
We’ve got a Dixon experiment, and then [00:17:00] on Wednesday, October the 6th, Gordon Dixon knew serum. I needed some good serum for later tests, so I got some from Gordon Dixon. And it’s labeled “later” in crayon as one with a circle around it. That’s when I began to call that pattern the “type one pattern.” That’s a later entry, but here is a Gordon Dixon serum made October 6th. October 12th, a George [Connell?] sample taken. So I’ve got good samples from my two friends, Gordon Dixon and George Connell, both of whom are graduate students. [00:18:00] Charlie [Haynes’?] lab. And these two samples later become the record sample, as it were. So here is the George Connell serum on six volt per centimeter, and yesterday’s 15.5% gel. Mechanically, very good. This particular gel was not kept, as the October 14th one is much better at 16%. So I got an even better run.
October 14th, nothing very special. October 15th, Gordon Dixon serum and George Connell serum set up successfully in new trays. Then I have [00:19:00] an image of the two gels, which I always like to show if I give a talk about the work. So it’s page — subsequently labeled page 81 in the September 1954 book, where George Connell and Gordon Dixon samples are sketched. That page 81, I’ve used as a slide many times. It’s one of my favorite pages of the samples. And I always make the little joke, I got tired of getting blood from myself so I got blood from my friends. Partly true, but certainly from my friends, and I was a bit tired of giving blood. [00:20:00] (phone rings)
Then, on October 25th — Monday, October 25th — I got a sample of blood from Beth Wade. [00:21:00] A usual one hour room temperature to the cold at 1:00 PM, centrifuged, et cetera, and four observers can see no hemolysis. And I make the comment that this is Rh negative blood with double on the line, so I must’ve been wondering if there might be anything different in the Rh, but whether my — I could see any difference in the samples. The comment is, “Get more sample of Rh negative. Although the Rh negative is due to absence of antibody” — of course, that’s antigen, really, on red cells — “check if Beth Wade ever had transfusion or was in menstruation.” I don’t know why I was thinking about that at this point. Only 20% of Rh negatives develop antibody [00:22:00] to become Rh positive, ever should test quite a number. I think it’s probably because of at some time in that period, I began to be interested in blood groups and had gone to have — in part, to learn something about blood grouping. And by going to the hospital for sick children, where a very great friend was at the — in head of — was head of the blood group. Her name will come back to me in a moment (laughs). Getting at my age, they disappear occasionally. Her name was gone for the moment… she was the person who found not the Louis blood groups [00:23:00] no. The ones related to AIDS — maybe come back and add a comment later about that. They were important in understanding why some individuals are resistant to the AIDS virus, whereas other people are not.
Interviewer:
A new one. Yeah, OK. It’s started now.
OS:
On this page, October — Monday, October 25th, I make a note that I received a 10 milliliter sample of blood from Beth Wade. Her usual one hour room temperature to the cold at 1:00 PM, et cetera, centrifuge, and so on. Four observers can see no hemolysis. I then have a note that it’s Rh negative blood and doubly on the line. I really don’t quite know why at this point I was interested in Rh negative blood, but I think it was probably because I’d been, in general, interested in blood groups. I’d gone to the hospital for sick children and had a little course — a private course, you might say — that Mary [Cutbush?], [00:01:00] Mary Cutbush [Croxton?], with her husband’s name, had — gave me a little course in blood group determinations, which I enjoyed very much. So I knew quite a little bit about blood group, but why at this point I was interested in Rh negative, I’m not really sure. But anyway, I got a sample from Beth Wade, which turned out to be quite important but not yet — it’s not yet obvious to me why it’s important. So I got this blood from Beth Wade on October 25th. And then continuing, I say on October 26th that Beth Wade serum was prepared — BW serum was prepared, and [00:02:00] a sample was run at 12:30 PM on six volt per centimeter, 15% gel, et cetera, et cetera. At the bottom of the page, I say that the run was from 12:30 until six– from 12:30 PM to 6:30 PM, and I have a note that, “Most odd. Many extra components.” And there were two gels being run, and both were excellent mechanically. And on the left hand page there, I have a diagram of — a sketch of Beth Wade’s blood sample, which is really quite different because four or five extra bands in the gel running quite slowly, and the gel is a sort of series. [00:03:00] So I wondered, well what on earth was different about Beth Wade? The obvious thing was that she was a woman. And Gordon Dixon and myself, and George Connell whose blood we’ve been running many times, had a different pattern without these bands. And so I thought that it was the difference between man and woman. So I called the Beth Wade sample an “F sample” and type, but Gordon Dixon and George Connell and I had the “M sample”. I’ve decided that this was pretty important, anyway, so on October 27th I have a note, “Decided to photograph the October 26th Beth Wade gel and the October 6th Gordon Dixon [00:04:00] gel.” In those days, I didn’t have a camera in my lab, and I had to arrange to have photographs taken somewhere else. So that was the decision made at that point, was to photograph these. And sometime shortly thereafter, I have a sample from George Connell that was included in this photographic effort.
Looking back for the page where those two samples occur together. Let’s see if I — here, it might be a little bit further down. [00:05:00] You know, we’ve already passed those samples. I must’ve talked about them earlier in this recording. They were on page 81, where there is a very nice sketch of George Connell, G. Connell and Gordon Dixon’s gels. So that page 81 is what I call the “M pattern” and page [00:06:00] 97 is what I was then calling the “F pattern”. So here we have the possibility of male versus female, is now in the equation. Thursday, October 28th, I got samples from another female, Diana [Mitchner?] — serum sample taken. I made a note because I wasn’t sure if this was male or female, or whether it was due to the stage of menstruation, et cetera, next menstruation about November 18th. So I wasn’t too shy about asking these young people, my age people, about their status. And a comment that the sample from Diana Mitchner hadn’t clotted in one hour because of my super method of [00:07:00] preventing hemolysis, except in a few isolated places. Therefore, a trace of filter paper, dust was added, and mixed somewhat, and it clotted then by about an hour and three quarters later. It was always nice that I had to induce clotting.
So there’s a dynamic sample being run. A comment coming then, “Better repeat, plus or minus hemoglobin (laughs). Experiment to determine if the decrease in alpha-one one, as I called it, by hemoglobin, is at all related to the new bands.” Which in very shortly thereafter, I’m going to find they are indeed affected by hemoglobin because both [00:08:00] are haptoglobin. So a couple more samples here, November 1st from Jimmy — I think that’s my friend, Jimmy. His age is not in there, but we’ll see. No hemolysis, and from a female, Rh negative underline, Carol [Broadhurst?]. Next menstruation, November 15th, so I am doing those samples too. So there is here not an image of the gel, not a sketch of the gel. I’m finding I’m [00:09:00] keeping the gels and beginning to think about having them photographed so there are less sketches. But here is two bands of color were visible in a short time, after about 10 minutes. Looking at it, I can see — this is page 108 — I can see that the one band would be the hemoglobin — free hemoglobin — and the rather smeary band is about four millimeters. That would be, as I would later find out, the multiple components of that variety of haptoglobin, that had not yet resolved into separate bands. Six hours after, I say that the plus hemoglobin is exactly equivalent to the hemolyzed sample [00:10:00] I got of a female of 54 years. Therefore, can conclude that all present in that sample — that all the bands are present in that sample, too. Then I want to try the male serum again.
November 2nd is rather entertaining — Tuesday — because I remember the history of it quite well. Jimmy, by the way, was Jimmy [Bryant?] and he was a good friend. He was a technician in the production side of [Connaught?] labs, as was the next person I’m going to mention, who was Casey [Cook?]. He was also a technician, but they were both good friends and we were very easy together [00:11:00]. But anyway, I got a sample from Casey Cook and from Miss [Lewis?], who was the secretary of that part of the Connaught labs. She was rather a dominant lady. I got samples from them and ran them, and well sure enough — big surprise — Casey looked like the female type, the F type, and Miss Lewis, the rather masculine lady (laughs) looked like the male type! This really upset Casey, and we gave him a hard time of it. Suitable remarks, oh come on, let’s have a look (laughs) and so on. But he was upset enough to ask his brother to come and have his blood sample tested, and his brother was a pilot in the Canadian air force [00:12:00] and he flew into Toronto airport just so he could — of course, pilots could fly their airplanes quite easily. He flew into Toronto airport and he was the only person who gave a blood sample who fainted (laughter). So the pilot fainted! His brother was upset, so it was quite a history. Anyway, we had quite a bit of joy with these things. So as I called these samples the alpha-beta samples, then the alpha-betas there in this 14-day sample, but much lower than other ones. So I just wondered what was happening.
Now I got a sample again from George’s sister, Mary [00:13:00] Connell. She found out was type — what later became type one, the “male type” as it were, and that’s why we had never seen it before. Because unknown to me, the samples that I would repeatedly get from the hospital for sick children when I was working out that detail were almost always from her. She would just give some more blood. And then on November 3rd, Otto [Hiller?], who was my technician and the father of our current technician Sylvia Hiller, gave a blood sample and he was also what we called the F type. So it was clear that this has nothing to do with being male or female and the result was that we began to think about — or I began to think about it — as being a genetic [00:14:00] factor. And the question was, was it related to any blood group that was known? And so forth. So I have a note on Thursday, November 4th that to date, one female has no alpha-betas as I called them, and five females have the alpha-betas. In other words, one looks like the M pattern and five look like the F pattern. But two of the males have the F pattern and four have the M pattern. So I still called them F and M but they no longer meant male and female. Though fairly quickly, it became clear that the so-called F pattern really had two forms that were both multiple banned but slightly different. One, we later found — or quite rapidly found — was the heterozygote for the gene [00:15:00] responsible and the other was the homozygote. But anyways, here is Miss Lewis, 13-day sample and Maureen, 18-day sample.
Let me just (inaudible) — that it were — so another pair of people, a male and a female the same day. [Muriel?] and Otto — Mrs. Muriel B, versus Otto. At that time, I was just comparing males and females but it quickly became clear, as I said, that it didn’t segregate with gender. [00:16:00] And then I first began to see that — as I said — the multiple bands in the F pattern were not always the same. They had slightly different mobility and there was another difference. And so the F pattern became divided into F1 and F2, which were heterozygous and homozygous for the gene two, as we later called it.
Friday, November 5th, I got a new sample from George’s sister, Rh positive (inaudible) and compared it with Beth Wade’s sample. [00:17:00] Oh, I think I must’ve repeated one here because I was worried that the sample had got muzzled. And I got Miss Lewis again and tried her sample again. Maureen maybe, maybe it was Maureen Connell. Let’s stop at the moment. Let’s see if we can find a Maureen Connell — just hold. And so now I’m looking at Saturday, November the 6th, and we got another sample of blood from George’s sister, Mary Louise Connell and from Beth Wade, just to make sure that everything was OK. And I have a note then that, “MLC [00:18:00] — Mary Louise Connell — is equivalent to George Connell, is equivalent to Miss Lewis.” And Beth Wade, as before. So I’ve got the note now, “Query familial.” So I’m now thinking of it as being familial.
The following day, November 8th — Monday, November 8th — I now see that they have — the F type is two varieties because Casey is not the same as Otto and Otto is not the same as Muriel. Muriel is probably the same as Casey. It’s a rather complicated diagram, but Otto and DM — Diana Mitchner — are the same, and Casey and Muriel are different. And these are what later became the — what first we called, that’s F1 and F2 — and later became a type 2A [00:19:00] and type 2B. Haptoglobin 2,1 and haptoglobin 2,2. It’s quite clear that I know the difference but haven’t yet understood it. Casey’s alpha-betas are not equivalent to Otto’s alpha-betas. Otto’s are slower than Casey’s, so Otto is homozygote and Casey is heterozygote. That’s what I later found out, what I knew at that time.
So some more two persons, again. 10:15, L. [Mackerson?] and George Dixon, again. But that was here — oh, Joyce [Taylor?]. So I got some blood from Joyce Taylor [00:20:00] and Diana Mitchner, and was looking at those Tuesday, November 9 — it was a very exciting time. Charlie and that sample, et cetera, Gordon Dixon, and LM — whoever LM, I can’t remember who LM is. L. Mackerson, L. Mackerson. He must have been a volunteer. And here I have two more people, Thursday November 11th: Cliff [Harris?] and [Glen Evans?]. He’s about 40 years old. For some reason, I put that in. I don’t know why. And his sample had to be seeded; it wouldn’t clot because of my precautions against hemolysis. [00:21:00] And here I was pleased, good enough for a sample photograph, underlined. Here I am, group A, the Rh positive grades, et cetera, et cetera. And so I kept record of where I could get it — of the Rh blood groups and the AB, O blood groups, where I could find them. I’m beginning to understand that these serum differences were independent of the known blood groups. And for the first time, on Saturday, November 13th, I began to call one of the patterns F1 and — no, I guess I had already begun on the 12th so I have to go back to the book there and revise that.
Thursday, November 11th, as I mentioned I had Cliff Harris and Glen Evans had to be seeded. [00:22:00] And then on the next two pages, it was quite a remarkable record, really. It’s every sample that I studied in this particular study is recorded on these two pages, 57 — no, 127 and 128. Altogether, it looks like 57 people had been studied. What I sometimes show students is a photograph of these two pages and say, “Now you have a data file. You have one in your computer, but I bet that you won’t be able to produce your data file 60 years from now — that a hard copy, old notebook has all of the records and the blood samples taken 50 — 60 years ago.” [00:23:00] And showing that the male/female difference — it’s not significantly different. The blood groups are not related and my F1, F2, and M patterns are not related to male or female and the blood groups is not related to the no [name viogler?] groups. It’s not related to Rh blood groups. So this is a document of knowing that these groups are independent of the red cell blood groups and independent of being male or female.
I didn’t at that time know what the protein was called. I knew it was hemoglobin-binding. Then I went to give a talk at a blood group meeting or somewhere [00:24:00] I don’t remember exactly where. Maybe I did just my publication of these observations on there was a hemoglobin-binding protein. And I had a message from [Laurell?]. [Avida Bethel?] Laurell. I think it was from her saying that, yes, well the proteins you’re talking about are already known and they are called haptoglobin. Hapto meaning “I bind” and globin. So these were hemoglobin-binding proteins that had been described by Max [Ferdinand?] Jayle, J-A-Y-L-E. In fact, he had even noticed that there were two forms — at least he could detect two forms of the haptoglobins — because sometimes they were in the urine, and sometimes they were not in the urine. [00:25:00] He could see the difference. So he was very close to finding that haptoglobin had inhabited differences, although he didn’t actually find it. He knew there was something different about haptoglobin not genetic.
He had rather a sad history because his method of quantitating or measuring the amount of haptoglobin consisted in using ethyl peroxide, which would in the presence of benzidine would give an intense color of developing — oxidizing the benzidine to a colored compound and was catalyzed by a hemoglobin-haptoglobin complex. He found the conditions [00:26:00] where hemoglobin would not give that reaction, but hemoglobin when bound to haptoglobin would give the reaction. I think it was a matter of pH, that at the high pH or the — I think it was low pH, actually — at the low pH, hemoglobin was denatured and would not give a benzidine reaction but hemoglobin-haptoglobin could withstand the low pH and therefore gave a reaction. But ethylene peroxide was a very dangerous chemical. He had an explosion with it — it exploded and he was blinded by it. So he was blinded by the acid that he developed for haptoglobin. And George Connell, later on, when we began to do chemistry that I’ll be talking about later — devised a method to use hydrogen peroxide, not ethyl [00:27:00] peroxide, instead of hydrogen peroxide which is much safer.
The story’s quite long, really, related to Jayle because he published all was in French in the [contron du l’ paller?] — I don’t remember if it was in Paris, but it was a French journal. And so I mean I had found it alright in the French journal. I didn’t have any problem with French because of high school, but it was quite a long time after he died. I should think perhaps — about maybe five years ago I had a telephone call from the son of Max Ferdinand Jayle saying that he was — that nobody ever took any notice of his father’s work because it was published in French. And I said, “No that’s not true!” [00:28:00] I said I knew very well about his work, with the help of Laurell to get me started. I knew very well about his father’s work, and the fact that it was published in French didn’t make any difference. So his son was then himself not a young man enjoyed this story very much, that we — I was almost sad that I never met Jayle himself. I would’ve loved to have met him. And we didn’t even correspond, so I don’t know whether he knew of the haptoglobin work or not. But anyway, Max Ferdinand Jayle is a treasured memory for me.
Going on with the story, Friday, November 12th, more people. Continuing that way, accumulating more and more people. So all those are listed on page [00:29:00] 127 and 128. They’re always recorded on that page but they’re actually studied later on — this study won’t come for quite a long time, collecting all these samples. Many of the people whose names I don’t quite remember any more, but they’re written down here. Shirley [Mooder?], Julie [Bonneman?] — I must’ve been able to talk people into giving me blood (laughter) because there are lots of people but I don’t remember their — who they are anymore.
Now talking about F2 and F1. 12:20 PM, Friday, November 12th, JB and SM, F1. [00:30:00] I think the other is probably F2. It’s written as a seven — JB looks like seven subscript two, but I think it’s probably really F subscript two, and SM has F subscript 1 which is correct. Beginning to think it might be possible to stain the gels so that they’re no longer precipitated. The starch was precipitated in the staining solution that we used, and therefore was — the images were all of a blue against a white background, as it were. And you only saw the surface stain, so even if it was possible to stain all the way, keep the gel — transport the element and stain all the depths of the protein, [00:31:00] one would be much more sensitive. And in fact, we did work this out and usually — occasionally, but it was more work than it was worth most of the time. But anyway, this was the beginning of that thought, the test of right through staining. I slice this test strip so I’m 1.5 to 2 millimeters and 0.5 to 1 millimeter, and left them overnight in the same. Dye was completely through by the morning, but I now wanted to try to make them transparent so that they were transparent. No word there on how to do it, although we did find out how to do it later on. (phone rings)
[00:32:00] Here’s a one Mrs. [Pulick?]. I don’t know why I called here Mrs. Pulick because the Pulick family are what became really great friends. As much as it were later on, I did work with Dave Pulick and his wife, who was a great friend. So Dave Pulick and Mrs. Pulick. (phone rings) I have a comment here, “In view of the through staining results and near transparency of one millimeter slices of the untreated gel, [00:33:00] decided to try the less drastic clarifiers again.” One was a saturated barium chloride, looked good on previous tests. Sodium chloride and magnesium chloride were possible with fully saturated solutions. So sodium chloride had very little clearing, barium chloride a little clearing. Magnesium chloride coming in quite clear, and calcium chloride — as usual — very rapid clearing. Conditions of the gels and stain, sodium chloride, color good, gel excellent, moderate clearing. Barium chloride, color possibly decreased a bit, excellent gel, fair to good clearing. Magnesium chloride, the color disappears [00:34:00] and the gel dissolves, and calcium chloride, the gel dissolves.Some [experiments?] here, the purpose of which escapes me at this point, were trying to look at the migration in the gel of dyes — a permanent blue back — oh, is it off?
I:
No, it’s on.
OS:
— a permanent blue/black and a medial black and sodium [picrate?] and [nasolin?]. So sodium picrate is well separated, but must use another two much more dilute — ink is no good. Why I wanted to study dyes, I don’t really know at that point. But I have a gel then. On the next day, Wednesday 24th [00:35:00] whether vital red or [medial?] black and sodium picrate are three dyes that would migrate in the gel. And here are samples from Mrs. Pulick. Mrs. Pulick, of course, being Emily Pulick. (laughs)
A new brew of dye mixtures. I’m trying to see, I think, if they made any difference to the voltage gradient. Beautiful electrophoresis, as it could, preliminary separation in 40 minutes but the medial black trails somewhat. [00:36:00] Stepped up the voltage, et cetera, et cetera. And then the training — trailing of the medial black didn’t migrate at a straight forward band. So, what that meant, I don’t know at this point.
More samples from different people — November 30th, Mrs. [Miller?] and Dr. [McVickers?]. Quantitative new tests of saturation — saturated solutions, for clarification. Strontium nitrate and cadmium chloride, sodium nitrate, sodium bromide, sodium sulfate, there is comments. [00:37:00] Potassium bromide good and rapid, but definitely not quite clear and three-quarters saturated sodium bromide very good, but possibly not quite clear. Saturated sodium bromide in difference in clarity not appreciable. But however, I have a comment that, “Considerable color washes out overnight and they may not be — may have to use short times.” I didn’t really like it.
More blood samples, December 1st. And December 1st, December 2nd, more blood samples. I would do two a day. December 3rd, two more. December 4th, Saturday, running two gel GI and AC. [00:38:00] And here is on the next page, on Saturday, December 4th, a chi-squared test of whether there’s any significance between male and female and their three types, which I’m calling 1, 2A, and 2B, or as F1 and F2 but I’m now calling them 1, 2A, and 2B, versus male and female of which I had 10 samples of 1 and 21 samples of type 2A and 12 samples of type 2B — which is interesting because they are almost in the natural 1:2:1 ratio of a homozygote, heterozygote, and the other homozygote. Chi-square was [00:39:00] 3.168, N=2, the p is approximately equal to 0.2 — that is, there’s one chance in five of getting these differences for M and F on some overall distribution for each sex, on the same overall distribution for each sex. In other words, gender didn’t have anything to do with the distribution of the blood group — of the serum group.
A note on page 181 [00:40:00] with a pathological sample, a sample from a female with carcinoma of the cervix and another female with carcinoma — three people with carcinoma of the cervix and one female with a primary stomach cancer and ovarian, secondary. So seriously ill people, stage one, late stage two — stage two with metastasis, though I never really did much work with the pathological samples. Dave Pulick was really — took over when looking at pathology. I found myself interested primarily in variations in normal individuals, and it had nothing to do [00:41:00] with being sick or not. And though there are no longer any diagrams of these samples because by now I’m recording them on film, and yet have not been making copies yet of the photographs. So the notebook is less satisfactory than was earlier, where there were sketches of the results and as it became later when there were photographs of the results. So this intermediate period, all the results are in microfile — microfilm, microfile — films that are too difficult to decipher at this point — not worth it, it would be [00:42:00] a waste of effort.
And here is an individual with primary ovarian and secondary — stomach primary and ovarian secondary, and that person relates stage two carcinoma of the cervix — but not really very different that I can see in this as far as these notes are concerned. [00:43:00] We begin to think about using other sorts of stains besides protein stains and mucopolysaccharides stayed in the fat stain, [twos?] and four in the new dye. Flavianic acid from [Fisher?] chemical company and Oil Red O — which later on was used very, very much for staining gels and for other purposes. It had been used for a long time for staining lipids.
On December 21st, I have down the comment that they’re 1, 2, 3 [00:44:00], 4, 5, 6, 7, 8, 9, 10, 11, 12 — 12 samples were run on that day and in a big circle or — on the page opposite, page 199, it says, “This completes all crosschecks. OK.” So I’m ready to publish the results at this point. So this pretty well concludes this book, looking through a little bit further. Flavianic acid didn’t work very well. Tribomophenol blue, didn’t like it all that much. Methyl red, cresol red, looking whether lipids ran in the gels. [00:45:00] Oil Red O, but very poorly soluble in 50% ethanol. I used to spend a lot of time looking at the dye — a good book I had on the dyes that were available, I’ll find out what that book is in a moment, just somewhere I can write it down. It was a book I enjoyed very much. I’m just going to find the beginning — don’t — I was, of course, very interested in dyes and because of their ability to stain various things and I had a marvelous book of biological stains published in [00:46:00] 1953, which I used to help people find different dyes. I think in there was probably where we first found wool [fast?] blue, which has a different name. It is used all the time in staining gel, to this dye. Let me just see if I can find it.
I had a book which was in a sense my “Bible of stains” — A handbook on the nature and uses of the dyes employed in the biological laboratory, by H.J. Conn. [00:47:00] I have a copy of the sixth edition which was in 1953, and there are many dyes because I was constantly interested in finding dyes that would give us better results. In fact, wool [fast?] blue BL, which is now generally known as Coomassie blue — I’m not sure, but it’s quite possible that Otto Hiller and I were the first people to use that dye. But anyway, it’s used very generally and Otto used to help make that — and I’ll talk about Otto Hiller a bit later on as we move through the books. But the dye book was a very important book in my library.
I’m trying to find the name of the person who said haptoglobin to me… I just in looking for this book on the biological stains, I came across another little book that comes from a period of time, by H. Laurell –on electrophoresis of human serum — sera at pH four to six. And that’s Helga — I think it was Helga Laurell — and it may be she and not Carl-Bertil Laurell who talked to me about [00:49:00] haptoglobin because I see that she has a reference in this book. Oh no, it was not her actually, it was her husband — Laurell, C.B. and Nyman, 1957, “Studies on the serum haptoglobin levels,” so that’s later than I’m talking about but it was one of the Laurells who made me aware of the protein we were working with was haptoglobin.
So here we go, we’re almost at the end of the Book Four. [00:50:00] (laughs) Here’s a not untypical Saturday, January the 1st, working just the same. Still worrying about the front and back of albumin, but I don’t know why I’m still worrying about it. Page 215 and 216, another rather more brief measurement of voltage gradients and sharpness of the back of albumin and the front of albumin. A rather nice diagram on page 218 of the decrease in voltage gradient [00:51:00] that leads — is followed by an increase — which leads to the sharpening of the back of albumin, the decrease leading to a sharpening because if the protein fell behind, it would need a higher voltage gradient and then the front having the opposite effect. That if you got — if you fell behind, you would get a lower voltage gradient, and therefore be spread out. But a sigmoid curve explaining — well, accounting for — the spread of albumin, but still to this day I don’t really know exactly what was happening. It’s probably an interaction between borate and starch, which is well known that the two [00:52:00] that the hydroxyl groups of starch interact with the adjacent hydroxyls of boric acid, some complicated interaction which I’ve never completely understood.
And so at the end of this book, I talk about Charlie [Holliwell?] and getting a blood sample from him, and that takes me back to the front of the book where January 10th — the last two pages are January 5th, and it was the front of the book is January 10th where mother and father have the great family and the two children, [name redacted] and [name redacted] — who I think, if I remember rightly, are twins. Let me just see if that’s the case. [00:53:00] Yes, they’re twins. Again, absolutely indistinguishable. It looks like one sample. They must’ve been identical twins, I think, with an F type of alpha one but no alpha-betas visible, very good electrophoresis, et cetera, et cetera. Every little detail is the same but there is no sketch of it, unfortunately, to look at at this stage. The father was F2 and the mother was F1, and the two children were not [00:54:00] — it doesn’t say whether they were F1 or F2. They could’ve been either with those parents. That’s the end of that book, and that we’ve stopped today.
I:
All right.
OS:
Got quite a lot done!
I:
Well you’ve —