Oliver Smithies:[00:00:00] On page, we’re talking now about little — lowercase a Book, April 4, 1969, going through to March, so it’s rather a short time to complete a book, but there’s a lot of [appendices?] in it, and continuing the task of getting these peptides from, and doing sequencing of 31 different protein preparations that I mentioned. It’s starting with bromine water and [Dylan?] 63 proteins, and [Carl Levin?] did the experiment. Gel [00:01:00] on the following page.
Bromine and hydrogen peroxide, and performic acid on page 5, trying to get some useful separations. They don’t look very good on hemoglobin, bromine and performic acid tests, see her book, or see [McAl?], and then some gels which are very unsatisfying to look at now, and with a comment that that was a Tris-borate versene gel, and it would be better to repeat with an acid urea gel. Learning how to hydrolyze PTH derivatives with a little equation as to what one hopes [00:02:00] to happen.
A rather impossible-looking chromatogram on page 12, showing what, we didn’t have the most sophisticated methods. And with page 13, consider therefore alkylation of S, presumably that means [sustain?] to prevent re-closure of the ring. Also try a higher pH, so trying to get a good method for hydrolyzing the thihydantoin. [00:03:00]
Continuing for several more pages of the same type. Conclusions again on page 23, some reaction scheme. (inaudible) test the following on page 25, “Treat the BDHs with iodoacetamide and sodium hydroxide, and then acidify with acetic acid,” etc.
I haven’t yet got a procedure that is good on the following page, says that this isn’t any good, because on page 27, circled in large, is “Didn’t help.” [00:04:00]
Light chains 27700 on page 29, this [esteomine?] derivative with performic acid. And, so on with the protein chemistry, such as it was in those days. Tests of trifluoroacetic anhydride in the book, the peptides, trying to, as a method of getting a modification of an amino group. [00:05:00] Still trying to get a good hydrolysis method.
Interacting clearly with David Gibson, (inaudible) here for example is, a statement on page 43, Monday, April 28th, PTH cleanup. Dave has used [extractants?] to [remove?] the PTHs in the presence of peptides, and decided to try something of that type. [00:06:00]
Provisional conclusions on runs 35, 38 and 41, of [Dylan?] and so on. These must be peptide runs. I don’t think they were synchronated runs at that time. They are, the type of thing that is illustrated in the paper by Dave Gibson, [Mikhail Levin?] and myself, on Figure 2, which shows four tryptic peptide chromatograms of the light chains on the [Technicon?] P-resin, so I suspect these are comments really on experiments that Dave is doing [00:07:00] with peptide analysis. I guess these are definitely not synchronated runs; these are peptide chromatograms. Run 45, more comments on these different runs.
Provisional conclusion on May 12th for runs 41, 42, 45, 46, 47, [00:08:00] and 48, in order to get aspartic and glutamic acid, only direct dry works; there were three methods being looked at. We were using manual degradation at this time, I’m fairly sure, that that’s also commented on in the Gibson paper, materials and methods. [Edman?] degradation, a manual method, was employed. [00:09:00]
More work on these peptides. Recent experiments from the sequenate being talked about on August the 5th, Tuesday. Hydroiodic acid, HI, unstabilized, with DTT in the sample gives various yields of thioxazolidone, a derivative of serine, goes to alanine about 75%; and threonine goes about 100%, AB and methionine doesn’t change at all. So, beginning to settle on hydroiodic acid. [00:10:00]
Oxidation talked about on page 69. Since the cysteamine derivatives have gone so poorly, yet oxygen is an active poison, decided to try, etc. Oxidation tests.[00:11:00] More columns, etc., gradients again, October 29th. Look at BALC mice for some reason on Monday, January 19th. The mice were born on November 29th. EHF — I think I might be (inaudible) — I have to find out that, so it’s talking about “EHF”; I don’t know what that means anymore. [00:12:00]
I had some sort of a dream on Tuesday, February 3rd, “Aim to use a toxin to selectively kill non-antibody producers.” Idea from talks with Dave on my suggestion of selecting methionine-containing, or non-methionine-containing, (inaudible) mouse myeloma. Dave suggested [a toxin?] or versus, so we thought about the toxin from cluster (inaudible), which were hydrolyzed as light red blood cells, but not lymphocytes. [00:13:00] An old batch which I must have had around, oddly enough. Tried to clean it up.
“Too old, abandon,” page 85. Trying cephalotoxin, alpha toxin. [00:14:00]
Unhappy transit of some material from Albert Einstein from [Phil Cafino?] on page 89, two vials of NPC-11, etc., one broken, one not sealed and leaking. Something about selecting antibody producers. [Bates’?] recovery yield of, etc., and then page 95, myeloma cells [00:15:00] from George; I don’t know which George that is, but I do remember seeing these myeloma cells in tissue culture. Oh no, I’m thinking of melanoma, not myeloma.
Another selection idea on page 99, a protein from [Harold Deutsch?], Philip’s Bench-Jones protein. Later on, I got a very interesting one from Harold. [00:16:00]
Cleaning up bet Bench-Jones protein, myeloma, see also [Y- 49?], just continuing the same sort of idea. A possible light chain deletion, which may be one that led to a rather interesting paper from Harold Deutsch, possible light chain deletion now [zig?] [bicloma?] myeloma, etc. Labeled [A Z?], CQ275, plus DTT, A15, A114. [00:17:00] Eventually, we did find deletions in the light chains. Look at that paper, which I have in front of me, with, this was with myself, David Gibson, and Ellen [Fanning?], and Percy [Parr?] and [Connell?] from Toronto, so we did eventually find some deletions in immunoglobulin polypeptide chain, and this must have been an early one, or this was from Harold Deutsch, and [00:18:00] he isn’t an author on the paper, but perhaps we used some of his material as well. It may become clear later on. That paper I’m referring to on the deletions was published in ’71, and we were only on ’69 here.
And then here’s the beginning of what eventually turned out to be quite an interesting protein. I got from Dave [Pulika?] a beta-2, as he called it, microglobulin, from sequence [00:19:00] 318, the rest of the sample was free (inaudible), what SEQ318 is, I am not at this point, clear.
Carboxymethyl cysteamine derivatives, I think, for cysteine recovery, were commented on page 119.
And then I tried experiments on 124, performic acid on insulin with a comment that it’s Lois Kitze’s birthday. [00:20:00] I’m purifying Crawford’s HCD protein. Whether that’s “Heavy Chain Disease,” or not, we’ll see. Cleaning up more (inaudible) proteins, really quite a nice chromatogram here. Much neater type of record than some of the earlier ones. 0.8 mLs of A102 was available approximately 8mg and put onto a column. [00:21:00] G25 Sephadex [beads?].
Here we go cleaning up proteins again, Crawford again on page 133. Continuing, here’s a dog beta-2 microglobulin. This will be eventually interesting I’m sure, [00:22:00] because I remember the names of the dog. This is Friday, January the 14th, dog beta-2 microglobulin. Clotter’s blood from sibling MLC, major histocompatibility light chain, I think MLC, or I forgot what MLC stands for me, identical dogs Mary, and [Yum-yum?]. These two dogs have about a three-year-old kidney graft, and have had no immune suppression after the first six month samples from Dave Poulik, MDP, for beta-2 microglobulin, Mary and Yum-yum, urines were being freeze-dried. [00:23:00]
Crawford, again, again, again, on page 141. So we go, purifying these different proteins.
Mary, urine — dog Mary, that is, on Saturday, January 22nd. [Tipper?] is the one that eventually was interesting, or Tip; we’ll probably come across him a little later. Trying to make a film of collodion [00:24:00] for no obvious reason, a membrane. Trying to make a membrane through which proteins could diffuse, and that is how Book a ends, making membranes.