Oliver Smithies:
[00:00:00] This is Book b, little b, starting March 18th, 1972, and going through to October, so it’s about a six month book. Starting with Saturday, March 18th, a large scale dog urinary protein, making a fairly big column, intending to take about 10g of urinary protein in 100mL of lithium iodosalicylate buffer, and Sephadex against 0.1M sodium bicarbonate, to get rid of absorbed material, and then freeze-dry it and re-chromatograph it, quite a sensible procedure. And here is the, [00:01:00] on the following pages, the result of the column.And, gel on the material on March 23rd, Thursday, page five, showing nice, single banded protein. Then, most likely, maybe that’s not the same one, I need to check. That isn’t the same one. It’s back to (inaudible) a little bit. I’ll start again.
Starting again on Book b, [00:02:00] begins March 18th, 1972 and goes through October 27th, and it’s beginning on Saturday, March 18th, talking about intentions of making, and how to make or prepare large scale of dog urinary proteins, and what I intended to do. Looks as if I may have done this, because the intention is to take 10g of protein, etc., etc., freeze in a chromatograph, and at the bottom, there’s an entry that 6.7g of urinary proteins plus 50mLs of distilled water, etc., centrifuge for 10 minutes, and gave a dark brown clear supernatant, which is the material [00:03:00] that I then added iodosalicylic acid to it, and ran a column, and the column results are shown. The spot tests of chromatogram, where fractions 11-15 have a lot of protein.
The Thursday, March 23rd, is a different experiment. This is a G-100 column, cleaning up g-gamma light and heavy chain; the various fractions collected and labeled with Roman numerals. The column, which is page 7 and 6, [00:04:00] find two pages, the same fractions 1-7, or 1-10 (I-X) Roman fractions, freeze-dried, and run on a TBV gel, where everyone can see the starting material, and some earlier fractions, and fairly substantial amount, and not very impure of the protein that was from the urine. [00:05:00]
And next page is Friday March 31st, and results from VII from page B7 show no significant sequence, because of excessive salt, so dissolved, etc., and cleaned up with a mixed-bed resin to get rid of the salts. And, a little note to Dave Gibson about what you might expect from the different fractions.
Goat colostrum from [Kent?], a quarter of a total sample to MDP [00:06:00], that’s Dave Poulik. Aha, here is Tip, urine collection [step?]. This is the dog that I remember. So this is page 11 of Book b on April 3rd, and Dr. [Malaka?] suggest that the dogs which have had 6,000 rads of radiation before their kidneys went into failure might be worth a test, and there are two mentioned here on this page, Tip and Tim, which were the cages, and looking at it, there’s a note about Tim in very poor shape, and no urine, and the animal was sacrificed because it was — not by me, but by the people doing the experiment, but Tip was there. And urine [00:07:00] collection was started, so this is, I used to go every night and collect the urine from Tip for quite a long time. Here he is on page 13. Tip was transplanted March 27th, and so here we are in May, about a month later. No, two months later, aren’t we. So I have two samples of urine from him that I’m talking about, Tip-1, and Tip-2, etc. And got 3.2g from one and 8g from the other, with exclamation marks, so a lot of protein was being obtained from Tip. And some charts from Tip-1 and Tip-2 were sent to me, by [00:08:00] the people in [Thormund?] Hospital, that Dave Poulik must have been doing. So I got now three samples of urine, Tip-1, Tip-2, and Tip-3. Oh no, Tip-4 looks better, and Tip-5 even better, 1.62g.
Chicken [AlBC/ABC?] nuclei. This must have been in relation to getting some histones, perhaps, maybe in relation to Michael Sung’s work, we’ll see. No, it wouldn’t be Michael Sung; [00:09:00] it’s too late for Michael. Separating nuclei from this sample, for example on page 21, see nuclei radially dispersed, but dirty, query two populations, trying to get the nuclei. So, with an asterisk on Saturday May 27th, page 23, I tested the nuclei from B21 (dirty) in water. No solution, in water plus borate buffer, clearing, but just due to swelling, etc., etc. [00:10:00] And, with distilled water nuclei with borate buffer, marked increase in viscosity. Looks worth more effort, that the DNA is beginning to be liberated from the nuclei.
More experiments, trying to disperse [00:11:00] the proteins from the nuclei. Not very clear. pH effect on 0.1M lithium iodosalicylate, Friday June 9th, tested in the ultracentrifuge, or planned to test. But, conclusion from a gel run on this material, page 31, that this lithium iodosalicylate, even on pH 9.4, does not dissociate the DNA histone. Maybe this was at a time, I remember having a hypothesis that histones might serve [00:12:00] as a memory from one generation to another, since histones are transferred — some histones are transferred from the germ, and wondering, I remember writing a sort of paper on it, but I don’t think I ever published it, on the hypothesis at that time. Most of the pH tests, of lithium iodosalicylate with pH 9.3. Continuing with this work.
So, moving away from the immunoglobulins towards looking at histones. Overnight ultracentrifuge on page 37 shows no histone in any supernatant [00:13:00] from 0.2M [less?] and some or all gel pellets. Testing at different pHs, on the following pages. Tempting to centrifuge all these on different cushions, presumably, formula iodosalicylate. Therefore, test phenol and salicylic acid, [00:14:00] without the iodo. And the result on page 41, benzoic acid negative, phenol negative, salicylic acid negative, all with high pHs. “Try di-iodophenol, and di-iodobenzoic.” My conclusion on page 42 shows that negative results are dangerous, suggest that benzoate alone fetches something off, but also suggests that lithium iodosal (inaudible) at pH and benzoate at pH 11, nuclear that are of their equivalent.
Continuing to [00:15:00] mess around with the system, and not seeing much progress. Back to urine, and here is a picture of Tip, ah, good, a picture of Tip. Dog urine, Tip, Tip-9, 1.5g, and a bulk of 5, which is 5.3L, 9.8g, and on Thursday, June 22nd, Tip beta-2 microglobulin is identified as the equivalent of human beta-2 microglobulin, and eventually, this beta-2 microglobulin sequence was published. There’s a photograph of Tip lying down in the cage, and looking out from the cage, and being petted. [00:16:00] Whether by me, I don’t know. But 10.4g of this protein, Tip (inaudible). I sent the material to Poulik.
Frank V is another dog. Lots of urine. Threw some away, but this is not my writing, although it discontinued. Discontinued temporarily on Friday, July 21st, seems, essentially [00:17:00] negative, and the dog is awkward to handle, replaced by dog Sue. Sue gave 1.24g. Subsequent day’s urine volume much decrease.
So by page 57, “0.25g! only, very little protein after being used to Tip.” But on Tuesday, Wednesday, August 16, urine back to 1.5L. And the animal man says, no water. Upset, but just back [00:18:00] from vacation; perhaps not watered properly between times. So, but however, 2L of Sue’s urine, and another liter the following day have 1.7g and 0.9g altogether. I managed to get 7g from Sue.
James, a new dog; so here we are doing many different dogs.
Some immunoelectrophoresis for Lois Kitze on gamma-globulin on September 7th. [00:19:00] Separating material, [A?]477G, NS, I think, “normal serum,” and anti-J sample, dialyzed, etc., run on a column, and a gel. But, why I’m handling this is not clear. Let’s look back and see if AJ was the same sample as on the previous page. [00:20:00] Not very clear.
Bovine gamma-globulin, A1 and A2 (inaudible) onto a column. This is anti-J, and normal serum. Different allotypes of bovine serum.
Continuing this on page 65. And [Harvey Favor?] who was around there at that time, and I was friendly with, and helping, and doing things together, ran immunoelectrophoresis on some of the material. [00:21:00] Quite nice immunoelectrophoresis, although I always had some difficulty interpreting them. Never felt very comfortable with them.
[Milstein’s?] method was talked about on September 18th, Monday, page 69, which is a gradient elution of column, (inaudible) sodium chloride stripped, etc., etc. This was anti-J, material DB477-G, [00:22:00] normal serum. Dave [McCain?], who came at that time, papain used to make FC and FAB, I don’t know whether this is the time when I heard of the [getting?] of the Nobel Prize being given for the FC fragment versus the variable fragment, that got the Nobel Prize for —I guess this is a rather entertaining story from my point of view. Beginning of using papain to separate the FC constant fraction of the FAB, the antibody-binding fraction that [00:23:00] Rodney Porter had described, and for which, very shortly after this date, he got the Nobel Prize, and I have a little exchange with him that I think I might find in this book, when we get to about October, September 19th at the moment, making — fractioning the constant and the heavy chain. Have a constant region, and the variable region of light chains, no, of immunoglobulins, rather, [IDG?].
Papain test again, September 21st. And, gels on the cysteine plus papain cysteine, plus DTT, plus papain, etc., on page 77. [00:24:00] But, the comment that “the proportion of a constant region relative to the FAB seems low, so use more protein and try again.”
So here was 195mLs on to dialyze, anti-J, DB, A1, A2, ready for use, and very good separation of the gamma-globulins, and it says no better, and possibly worse than Milstein, but I think the separation is really quite nice; this is just a separation of immunoglobulin from albumin and so on. [00:25:00] Looking at the gel, I would be quite pleased with it now, I don’t know how I was a little bit disappointed somehow, but a large-scale digest with a longer column, and adding papain on September 22nd.
Gels of material on page 83, and here we’re getting closer to the time when I think there’ll be the entertaining comment perhaps. Monday, September 25th, sequenated tests of cattle papain FCs. Gamma-1 papain tests on Monday, 25th. [00:26:00] There’s no document immediately available of what the sequenated test results were at this point. And a comment on tests again, that 89, that the proportions of Fab, and Fc, in this gamma-1 IgG seem much more reasonable, trying to look for the constant part of constant region.
Bulk gamma-1 digest, September 26th, and Fab recovery on the 27th. [00:27:00] With some comments of difficulties.
Thursday, September 28th, various comparisons, which I thought would be done. FAB and FC recovery on page 97, freeze-dried material. Longer-term, low-enzyme tests following Dave Gibson on the 30th. [00:28:00] Try solid enzyme on the 29th, Friday. Freeze-drying something here, with a comment that “30 minutes, found the [sphere?] too hard to touch easily, so 30 minutes may well be much too high a temperature, something wrong with the machinery.”
Brief room temperature papain tests on October 2nd, getting very close to when it will be entertaining, I hope.
October 3rd, repeat bulk digest of gamma-1. [00:29:00] And October 3rd, time series at room temperature. Still digesting with papain. And, tryptic digest of Fcs, constant region I presume, trypsin inhibitor, trying different length of digestion at 25.
High temperature trypsin Fc method on page 115, October 6th. October 6th, some more [00:30:00] work with a pool of good gamma-1, trypsin. I’m not finding what I expected here. I expected on October 10th, to find a comment here, which I’ll refer to in a minute. [00:31:00] I guess this is further down than I thought. I’ll go back to there again. I guess, I’ve been looking ahead to try to find something. So going back to page 127, repeat of pepsin and chymotrypsin digestions to try to get the constant fraction. Doesn’t look [00:32:00] very promising, again, but continuing this sort of work on 0 degrees trypsin gamma-2, and 0 degrees papain gamma-1, so the papain is the way that Rodney Porter used.
Going on with this work, October 12th, 131, room temperature trypsin with and without DTT, and a bulk papain at 0 degrees. And continuing this on page 133. [00:33:00] Results on October 12th again, result are just 12 hours at 0 degrees, 1/100 papain is not enough. But [14/40?] hours is quite safe for gamma-1, so stop at 24 hours. Trying to get a good separation of papain-digested material into the constant and variable parts.
Repeating the columns and the digests of A1, A2. More work of the same type on October 15th. October 17th, [00:34:00] similarly, continuation of 0 degrees bulk papain digest of gamma-2.
Purifying the Fc fragments on page 141. Mary’s urine [grain?], and Fab tryptophan cleavage tests on October 26th, and [00:35:00] continuing here with this type of experiment.
And here is the pages I was hoping to find, on Friday, October 27th, [A2A2?] material, saying, with a comment, “Recrystallization of Fc papain, gamma-2, 0 degrees, about 12mg of B-143 Fc precipitate, were dissolved, etc., and let set to dialyze in the cold room within less than an hour, crystallization had commenced, and in a nice, heavy, blue, magic marker, “Quite nice crystals, bigger [slower?], didn’t mix until Saturday.” [00:36:00] So this was the observ — that I had found making the material that it had crystallized on me, so, in a way, I found what was already known from Rodney Porter’s work; I’d seen it happen myself, and so I thought, by October 27th, that was after the Nobel Prize announcement had been made, and I sent a little telegram to Rodney Porter saying that I think I understood what had happened in his experiment, but he’d taken purified papain digest protein immunoglobulin, and had cleaved it, and then had dialyzed it, or whatever, and come back in the morning and found crystal, and that’s how he realized it was a constant region, but not sure whether that’s true, but attentiveness of the message, which I know from Nobel [00:37:00] experience that you get so many congratulatory messages, that you can’t reply to them all, and I never did hear from him, but I always enjoyed the thought that I’d seen what he had seen when he made his important discovery that crystal, so page 150 and 151 of Book b are particularly entertaining for me. Freeze-dried supernatant, 10mg, wash the crystal, one time, etc., and I think I recrystallized them. An enjoyable experiment.
Comparisons on the last page of this book, of the various Fc fragments which don’t all migrate the same [00:38:00] speed. These are 72 hours at 0 degrees, and the fractions that are obtained, which migrate at different places on the gel, so it isn’t — again, I’m going to go back and look at that again. These comparisons of gamma-2 constant fraction Fcs, etc. Those are from B-141 and go back and see what B-141 experiment was. And the B-141 experiment was [00:39:00] of A1, A2, so just looking at them, I think there are a whole bunch of different Fc parts. I’ve got Fc0, Fc1, Fc2, Fc3, Fc4 on the starting material, and the gel showing the constant region. It has a nice, sharp band, but depending on where you elute on the column, you get different products. And that’s the end of Book b, little b.
OS:
[00:00:00] This is an addition to Book b, October 27th, Friday, page 151. I looked up a copy of Rodney Porter’s description of his work on the finding of the constant region of immunoglobulins, and he said it very clearly, and I was aware of this, but not, of course, of his Nobel lecture. They’re talking about digesting with papain. “Return to papain digestion of rabbit gamma-globulin was made seven years later, but in place of the crude enzyme [00:01:00] used earlier, crystalline enzyme was used at a much lower concentration, 1/100 the weight of the substrate,” which is what I was using in my experiment, because I was following his work. “Under these conditions, a number of points missed previously became apparent,” etc., etc. “The products are all of very similar size, where one third of the original, rather than one quarter, and most surprising, one of the products of digestion crystallized very easily in diamond-shaped plates during dialysis, neutrality in the cold room.” That’s exactly what I had observed, that it was during, in the cold room. I’m not sure whether it was during dialysis, but after dialysis [00:02:00] that this is where it says that, “12mg of Fc precipitate dissolved with ease, and set to dialyzed against 50mLs of water in the cold room. Within less than an hour, crystallization had commenced,” almost exactly what he had described in there, and that’s why I wrote to him when I heard he got the Nobel Prize.