Oliver Smithies:[00:00:00] So this is Book J, goes from nineteen s‑‑ end of 1962 to the beginning of 1963 — and starts off with — on page 3 with attempts to improve agar packing and separations with spherical particles, trying to make spherical agar particles, by spraying 3.5% agar over a large dish. And then tested in columns. And trying to make, really, spherical particles of agar or hemoglobin agar that can be used for separating of [00:01:00] material. For example, on page nine I say, “Try also spraying polyvinyl chloride and dilute agar to get faster settling of gradable particles,” where the PVC would be the center and with an agar coating. Not clear why I was worried about this but trying to get an improved — what would now be an ordinary column — partition column — and trying to make them sediment more easily than the fine agar alone — beads. So with polyvinyl chloride center, they would separate more easily, as exemplified a little diagram on page [00:02:00] 11, Sunday, October 7. And then a test of the system, on page 15. Fractions from a fast-coagulated agar were collected and tested. And India ink was also added, to see what would happen. And this is with haptoglobin 2‑2. And (laughs) the result is, “No appreciable separation of the polymer.”
Remembering the work that I’d done with Michael Sung, [00:03:00] a long time ago, of separating histones, with rat liver nuclei isolated from sucrose centrifugation. (pause)
Yeah, I forgot the… (laughs) What happened? You can’t remember any…?
No, I’m trying to figure what was happening.
NM:[00:04:00] You are working with DNA no‑‑?
I’m recording, Nobuyo.
(laughs) No! I don’t know why the DNA… And I’m trying to figure out, on Tuesday, October 9, for no obvious reasons, I went back to making nuclei — and the method that Mike Sung and I had worked out a long time ago. And then taking the histones to… Very weird experiment, completely incomprehensible at this time. [00:05:00] (pause) Not at all clear why this weird diversion into histones — unless it was a thought that it might help understand how to purify…
Page 23, Wednesday, October 10th. This looks more straightforward. Two-point-five percent agar column, continue… And a column was made and then fractions were prepared. And the result, on [00:06:00] page 25 — it says, “Very nice but not different from usual separation of haptoglobin 2‑2 from impurities.” (pause)
So every now and then, trying to improve the photography. Filters for photography, on page 33, Saturday, October 20. (pause) Looking at the [00:07:00] spectrum of NRS ion, at different pHs, on page 35. Histones again, on October 22nd — not photograph. But why the histones were being done is still a little obscure.
Two-dimensional split, on Monday, October 22nd. And molecular weight, a more straightforward [00:08:00] experiment. On page 43, a straightforward molecular weight on ceruloplasmin, and from Dave Poulik, MDP. Ceruloplasmin was tested, with insulin B chain. And there were several components being studied. Insulin B chain was there, ceruloplasmin, alpha-ceruloplasmin, beta-ceruloplasmin, gamma, and ceruloplasmin-delta. Attempt to get these — the molecular [00:09:00] sizes.
And a new starch being considered, on page one‑forty‑‑ on page 45, low-background starch. Straightforward thought. But no change in background, is the conclusion. Washed and unwashed, in some special way. Myeloma protein from a person, Burnett. Gamma globulin being tested, on page 47. More t‑‑ starch background effects, on page 49. “No effect.”
Ah! Rough tests on various single-donor 2‑1s for [00:10:00] the final gel, to assure the — that the 2 is constant but the 1F and the 1S are different. But only really two components, the 2‑alpha and either 1F or 1S, not a double one. The gel is not a very good gel, on this particular day. A better gel on the following day, called, “Hp finals,” w‑‑ November 1st, Thursday, page 53. And I’ll go back and look at the final publication and see whether this is really the final gel. On page [00:11:00] 53, Thursday, November fir‑‑ the — what are called, “Hp final,” trying to get some final gels. I don’t remember whether these gels were ever published or not. But the diagrams of the gels were published, in “Gene Action in the Human Haptoglobins,” published in 1966, where the diagrams on page 216 of that publication are s‑‑ comparable to what — being seen in these gels.
Well, so we’re continuing to make — I’m continuing to make [00:12:00] sintered PVC, at this point, on page 55, to try to get some columns. Going on for several pages on making these different columns. Background — the starch again, page 67. Block any free NHQ groups, as usual, trying, for example, acetic anahydride, plus or minus pyridine or nitrous acid, acetic acid, etc. Later on, we did find a method. I don’t know whether we’ll come across it in this book. But that was just to add — my recollection, you add a little bit of heparin. [00:13:00]
And these are samples treated in — or gels treated in different ways, on page 69, although the photograph doesn’t appear to be of multiple gels. It’s a single large gel. (pause) J‑69. So the photograph is [00:14:00] of a gel, J‑69. But the gel diagram is of multiple gels, not of a single gel. So it’s not clear what the gel is referring to. (pause) Very clearly, the photograph says J‑69. And it’s of a multiple gel. [00:15:00] [00:16:00] (long pause)
A rather strange isolated photograph, on page 78, with nothing that corresponds to the same thing, rather like the one on page 68. Seventy-eight, these are two images with very little information about what they are. [00:17:00] Rather careless notation, I think, at that point.
But back on track on Thursday, February 21st, with an ordinary sample. But in this case, Mrs. [name redacted], Bence Jones protein, from the urine, being tried against her serum — with a comment that it’s clear that the single Bence Jones protein in the urine — “No myeloma protein visible. Confirms Dr. [Schilling’s?] report.” So the Bence Jones protein, you can find but you can’t find the myeloma protein in the — in the urine. Rather sad stories with [00:18:00] regard to the use of gels for determining the relationship of Bence Jones proteins to the myeloma proteins I may comment on later but not at this point. M‑‑ continue with Mrs. [name redacted]. Urine filtered, etc., on Friday, 22nd. Attempting to purify the Bence Jones proteins, on page 87, DEAE purification of the Bence Jones protein. And it looks very nice, the clear separation [00:19:00] between Bence Jones protein and what I think must be the myeloma. But let’s see. Pooled precipitation of dialyzed material was added to the column, etc., D– And elution commenced. And pooled the different fractions. Very clear that there were two components, a fast-migrating component and a slow-migrating component. Testing them in a TVV gel, on the following page. [00:20:00] And urea tests, urea, mercaptoethanol tests on the [name redacted] Bence Jones protein, showing the usual recover– “Used the recovered solution and set up a urea, mercaptoeth‑‑ and formic acid gel,” for the samples. Really very pretty electrophoresis on Friday, March 15th. Eight-[molar?] urea, 0.05-molar — 0.01 — formic acid, with 0.2‑molar mercaptoethanol. A very nice migration.
These urea, formic acid gel were, in fact, used by Poulik and Gerry Edel‑‑ by Dave Poulik and Gerry Edelman, [00:21:00] in initial tests of — that led to Gerry Edelman getting the Nobel Prize for recognizing myeloma proteins, light and heavy chains, and Bence Jones proteins, the light chains, and so on. They did acknowledge the use of my gels. They thanked me. But I think perhaps I ought to have been an author of the paper. Because they — it was dependent upon that gel that their results were obtained, the use of 8‑molar ureic a‑‑ urea, formic acid gel to separate the myeloma proteins and the Bence Jones proteins into their subunits.
Looking at trying to do fingerprints on these, page [00:22:00] 93. I don’t know why I had decided to do fingerprints but evidently began to think about doing them. And the various things one does to do a fingerprint. And the finger‑‑ first fingerprints that I obtained are on page 99, of material that had been prepared as a com‑‑ cu‑‑ set up for fingerp– And let’s go back. On Saturday, March 9, digests were made of the different preparations, [name redacted] impurity, etc., [00:23:00] etc., with… After digestion, mon‑‑ gels are run in one direction — in one dimension, on page 97. And then the fingerprints were set up, as shown on the bottom of page 97. And the resulting fingerprints are on page 98 and 99, with not — with no real comments on them. (pause) [00:24:00] Small-scale fingerprints, on Sunday, March 10, of freeze-dried — what do you call –? — A‑ and B‑grade [name redacted] Bence Jones proteins from J‑prime-89 — and for fingerprints. And the fingerprints are shown on page 106 and 107. What I was going to do with them is far from clear. But the fingerprints are really quite nice fingerprints. [00:25:00] (pause)
Trying to think about looking for — a [haptin?] search, Monday, March 25th. (pause) Thinking about the Gm groups and the Inv groups of — that [00:26:00] have been now begun to be understood in the inheritance of gamma globulin variants. Tested for inhibition of Gm(a+) and Gm(x+) and Inv(a+), for ve‑‑ for a slight — very slight indication of inhibition, etc., etc. So trying to get into this field of gamma globulin variations. Digestions of the hemoglobins. [00:27:00] (pause) And these are quite obscure tests, for me, at this time. But I think it is the beginning of my interest in gamma globulin variability. Because I did later publish a paper, a modern one, on — a hypothetical paper on what might be the cause of antibody variability. But I have difficulty in interpreting these [00:28:00] now, although it’s clear that I was investigating Gm variants and Inv, which were light-chain and heavy-chain variants of hemoglobin — of the immunoglobulins. Preparing plasma to make gamma globulin, on page 117. And we did a lot of experiments with purified gamma globulin, which no doubt I’ll come to in due course.
Could… Wednesday, April 3rd, page 121, a quite nice preparation of gamma globulin from serum. [00:29:00] Large-scale chymotryptic digest, following. Page 125, April 4. Clearly understood what was happening with Gm groups and the Inv groups. At least I understood the notation, because the sample is labeled as being Gm(a+), ‑(b-), ‑(x-), Inv(a-). And that was that particular sample of gamma globulin. And a larger-scale preparation on page 127. Here’s [00:30:00] Mrs. [name redacted], page 129, preparing Bence Jones proteins from her urine. Pooled the fractions for freeze-drying. And I have a note of 811 milligrams in the bottle, very nicely purified material. On page 128, a gel. New preparation, 20.07 liters of urine, deionized, and then adsorbed to [DEAE?] and eluted, April 9th, with a comment of, [00:31:00] “1,385 milligrams in the bottle.” Urea tests, on the [name redacted] and [name redacted] samples. Possibility that even crystallization was considered, on page 133. A gel was looked at, 8‑molar urea, formic acid, 0.5‑molar mercaptoethanol, good electrophoresis. Fingerprints on page [00:32:00] 135 — no, correction, a fingerprint and the chymotryptic d‑‑ gel first — so the gel and then a fingerprint. With a comment that the fingerprints show that J‑97 is now much more complex. Very complicated fingerprint. (pause) Fingerprints again, on page 140, on [00:33:00] Bevin and — [name redacted]. Eight‑‑ let’s — 80 — probably [name redacted] samples. [name redacted] seems more simple, page 140. [name redacted] again, on page 143. More urine, following days. (pause) And ending the book with still recovering dilute [name redacted] fractions, [00:34:00] on page 151, with a comment, “In the bottle are 939 milligrams total of [J‑151?], [name redacted].” And the end of this book. [00:34:15]
OS:[00:00:00] This is a correction, to Book J, page 19. On page 19, Tuesday, October 9th, I was beginning to do digests of haptoglobin alpha chain and also wanted to compare with histones. So there is a brief description of making histones again, and the way that I developed I developed with — or Mike Sung developed with me, when he was a graduate student. So this preparation of histones in the top part of page 19. And then, in the bottom part, there is a description of taking different amounts of haptoglobin alpha chains — or different mixtures of alpha chains, [00:01:00] the 2‑alpha chain digest and 1FS digest, and running those all on a gel. And it’s — the result is quite clear, that, in the 2, the junction peptide, J, is visible, although where that is referred to is on the next page, page 21, where it says that “J shows well in the 0.0501 formic acid. And about five or six more peptides.” So that that is looking at J. I don’t know where five or six more peptides can be seen. But I can at least see what was probably J, [00:02:00] the junction peptide. And I think I can see — I thought I could see the N‑ and the C‑terminal peptides. Also ran the sample on a different gel — or a different pair of gels, as shown on page 21, the left-hand two again being of the histones and the right-hand samples being the Hp alpha chains. No comment on them. That’s the end of the addition. [00:02:59]
OS:[00:00:00] An addition to page 36 and 37 in Book J. It’s — this is a continuation of looking at the histones, in different gels. But why I wanted to look at histones, again, at this point, is not at all clear. (laughs) And anyway, the comment says, on page 36, “Generally poor result. Must purify the nuclei first and use better extraction procedures.” Though why I was doing the histones is ag‑‑ still not clear. End of correction. [00:00:53]
OS:[00:00:00] An addition to page 109 in Book J, that there is some uncertainty about what the motivation of these experiments was, from the recorded data. But it appears, from page 109, marked Monday, March 25th, that the — my motivation at the time was to try to search for what the haptin was, that it distinguished the Gm genetic difference in the heavy chains of immunoglobulins, and the Inv marker, which was an — proved to be a single amino acid difference in the kappa light chains and converted [00:01:00] immunoglobulin type Inv to immunoglobulins that had kappa light chains. This appeared to be the motivation of these experiments and is, therefore, needed to understand what was going on after page 109. This idea was further confirmed by the fact that, on page 115, Tuesday, March 25th, I talk about haptins continued and looking for whether the antibody could be inhibited by different preparations, whether a tryptic digest or a chymotryptic digest would inhibit the reaction between Gm and the antibody — bloo‑‑ an immunoglobulin of type Gm and the antibody. [00:02:00] But the — where these samples and where this idea of inhibition came from is not clear in the — in the journal — in the — in the book. Just page 115 clearly sets out some ideas of inhibition. But it doesn’t describe how inhibition is measured — but continues, on page 117, with more work on purifying the immunoglobulins. (break in audio)
Another addition. Page 133, Wednesday, April 10th, was urea gel tests on the Bence Jones proteins from Mrs. [name redacted] and Mrs. [name redacted], with a comment that “They are so similar that non-identity must be [00:03:00] proven.” I don’t know that I really completely understood what was happening in immunoglobulin variability, at this time. And whether other people did, I’m not — I’m not sure. But [name redacted] and [name redacted] were two Bence Jones protein. And the fingerprints are talked about later. On — later, on page 139, tryptic digests of the Bence Jones, [name redacted] and [name redacted], are set up, with one-dimension electrophoresis first and on a gel, with anhydrous stain and [Pauli?] stain. And then the fingerprints are on the following page, 140, one presumably being [name redacted] and the other being [name redacted], [00:04:00] thought they are not labeled in the — in the book. [00:04:09]