Oliver Smithies:

This is Book X, beginning December 4th, 1967, and running through — not very long book in time — running through January 27.  And it’s largely related to purifying these light chains or derivatives of the same — starting with Dylan.  A lambda derivatives test here.  But each way was a medium-quality lambda chain derivative, I think, from what it says here.  But anyway, it’s talking about a lambda derivatives test.  And the light chains are good — concentrated light chains.  And from that myeloma [00:01:00] protein.  It’s — the reference to “H. [Way?], 0139‑B” is not immediately clear.  (pause)

Review of the derivatives, on December 6.  “Clear that Dylan can’t be used without better study of reduction.”  Decided to hold a detailed work on these reactions until kappa-lambda separation has been tested, to separate the light chains into the two varieties, kappa and lambda.  [00:02:00] First begun on December 7th, which is…  And, “Try by previous people with G‑25 sulfoethyl-Sephadex.”  And so I decided to test with sulfomethyl-cellulose, presumably because I didn’t have the other.  (pause)

Tried to repurify H. Way, on page 13, but lost by fraction collecting error.  Digest of H. Way — [00:03:00] looks — on Monday, December 11th.  Looks very complicated.  Comment on page 15, December 11, “Concentrate on using fully reduced light chain or Bence Jones, with [SSEtNH3+?] or SO side chains,” to recover the N‑terminal large peptide, from [HBJ‑3?] type, trying to get the N‑terminal peptide and the — simplify the whole procedure.

Derivatives of [00:04:00] Snyder, [MLB‑92?], December 12th, Tuesday.  More column.  Snyder SS repeat, Wednesday, December 13, etc., on the following page.  This was a poor decision.  (pause) Continuing results, quite poor, on page 27, blank tests, etc.  [00:05:00] Snyder again, column graphs.  Snyder.  Snyder again, with many different tracks.  (pause)

The method — that procedure that was — allowed me — the column setup allowed me to look at the alkaline ninhydrin peaks, the ninhydrin peaks.  And then at [00:06:00] different concentrations.  It must have been a relatively te‑‑ sophisticated column — look at three things at the same time — with the [Auto-Technicon?], that system.  Protein chemistry, continuing.  Pages and pages of graphs, and columns.  Longer running time.  Non-performic acid tests.  [00:07:00] (long pause)

These were columns that were run using the Technicon system, [00:08:00] with the bubbles, which would allow one to control — with air bubbles, which would allow one to control fraction behavior rather well.  The Tuesday, December 26 page, 63, gives an example of what one can do, that one could have cyanide coming in, with a — and a delay column, and then nitroprusside, so that things would happen at different times in the — in different parts of the column.  (pause) Pulse suppressors and delay coils, so on.  It was a — it was not an easy system to use but fairly [00:09:00] rewarding, that — their system of separating regions with bubbles still remains an important idea in my tools of chemistry, at the present time.  Loading my much better columns these days, I still separate fractions by — or materials by little bubbles of air.  (long pause)

[00:10:00] Long column, with Snyder, page 71.  (pause) Comment on Saturday, December 30th, “Standard gradients are essential.”  More columns, more columns.  (pause) [00:11:00] Confirming, on page 81, that “5‑mM DTT followed by 25‑mM cysteamine dihydrochloride is safe without waiting,” derivatizing by the disulfide interchange.  Just straightforward slogging away at trying to separate things with somewhat inadequate columns.  Use of sodium [00:12:00] boron hydride, on page 89, which I use continuously, these days, on [gold?].  (pause) Just moving on, columns and methods of trying to purify.  Using an Auto-Technicon gradient, che‑‑ taking the pH, on page 103.  And said it does get up to 9.55 — starting on page 3.  [00:13:00] Just different ways of trying purify the material, nothing very striking or particularly important, trying wetting agents.  Hm.  On page 115, “[Try it on Photo-Flo?].”  Acetone, methyl alcohol, ethyl alcohol.  But…

So a summary of the situation, on Tuesday, February 6, page 117.  “In view of the digest problems, must determine the [00:14:00] condition for the real [thing?].  Try the following,” and eight possible thing.  Light chains with SSEtNH+ and only one SH group being attacked and then cleavage, so that all of the cysteines and the disulfide bonds were cleaved too.  And so on.  And iodoacetic acid and iodoacetamide and ethylene amine sulfite and phosphothionate, all different light-chain derivatives.

[00:15:00] Light-chain digests beginning, on page 123, February 13th, Tuesday.  Twenty milligrams of each, where the internal disulfides are still intact and only the light-chain, heavy-chain SH is reduced or where everything is reduced — and all treated with cysteamine dihydrochloride — digested with TPCK trypsin.

Tests of Bence Jones proteins, gifts from Harold Deutsch — [00:16:00] on St. Valentine’s Day.  (pause) Tests of double digests, with trypsin TPCK.  (pause) With a comment that “The double digest is completely indistinguishable from the single one.  Therefore, stick with the single digest.”

[00:17:00] (pause) Review of the digest conditions, on Saturday, February 24, page 137.  “Review of the latest results leads to the following conditions.”  Just TPCK trypsin digest of cysteamine derivatives.  (pause)

Tried to concentrate the light chains by an Amicon [00:18:00] filter, on page 139.  “The yields are poor and time is slow.”  So I set up a cellulose-TMA — [trimethylamine-C — cellulose?], to purify the — my column.  But abandoned it, in the end.  Another way, on Tuesday, February 27th, tried.  Again, abandoned.  Sulfoethyl-cellulose — actually, sulfomethyl-cellulose tried on February 28th.  Recovered 17 milligrams of material.  [00:19:00] Concentration [in Bates?].  These are digest material.  And ending the book with repeating the digests.  So Book 152 is ended — sorry — Book X, page 152 — ended.