Oliver Smithies:
This is book W. Starts August 27th, 1967 and goes through December. Still working with light chains. Derivatizing them on the first page. And thinking about peptide methods. Obviously these experiments are aimed at understanding eventually the variability in light chains in relation to various hypotheses that I and other people had proposed. Many of them were driven by attempts to find ways of testing [00:01:00] the hypothesis for gamma globulin variability which I had published in that period of time. For example in 1967 I had a paper on antibody variability and the recombination between elements of an antibody gene pair may explain antibody variability. The paper on antibody variability was published in August or June of 1967 and this work that is being carried out in the latter part of 1967 is aimed at trying to get more data to help in [00:02:00] understanding antibody variability. So I was thinking about ways of testing this hypothesis by isolating light chains or myeloma proteins and understanding how the variability was determined genetically. So still working on peptide stain Saturday, August 2nd. [00:03:00] Made a new column. Saturday, September 9th, [G100?] with a way of keeping the liquid above the column constant. Two hundred milligrams of gamma globulin reduced and alkylated, 8 molar urea, etc. And then attempted to separate the protein.
[00:04:00] Sunday, September 10th, gamma globulin to gamma globulin light chains. With tracing from a gel, from a column. Now I have ability to run column with optical output and the light chains. Perfect application on this column on Monday, September 11th. [00:05:00] Pooled material W33. So trying to make light chains and continue and so forth. Gel of the results on page 36 where the gamma globulin is being purified and eluted from starting with whole plasma apparently. Purifying material. [00:06:00] Pooled and frozen. Quite nice-looking from the gel. Tuesday, September 19th. Must have been reasonably pleased with it. Isolating the gamma globulin. Light chain check on Monday, Tuesday, September 19th. Page 45, 8 molar urea. Concentrated gamma globulin with heavy and light chains and then what were light chains plus and light chains pure. W33 pure light chains look quite nice. [00:07:00] Did an experiment with Lois’s gamma globulins. Bovine serum. On the 21st of September.More light chain cleanup Thursday the 22nd. Pooled W49 light chain. These columns took a long time to run as judged by Thursday, September 21st entry. [00:08:00] Light chain cleanup. PR out by late p.m., i.e., about 36 hours or one and a half days from complete wash of the column. They were slow columns. PR is a dye but I don’t know what dye it was. A [dumpy?] column of [G75?] made. Run overnight. [00:09:00] Making light and heavy chain. Gamma. Friday, September 29th, gamma [G1-242182?] light plus heavy, etc. Concentrated and a big comment. Thrown out. [00:10:00] Cleaving the disulfide bonds between the light and heavy chain with guanidinium hydrochloride on October 2nd, Monday. Although column didn’t work very well. And so on and so forth continuing to separate light and heavy chain. [00:11:00] [00:12:00] Having some conclusions on page 85. Round about October 8th. Even at this stage it is obvious that reduction should be in the absence of urea. Therefore use 5 millimolar DTT. No urea. With [cysteamine?] dihydrochloride. Get a stable product. Cool and acidify and add solid urea to 5 molar. [00:13:00] [00:14:00] Lengthening the column on page 93, Saturday, October 14th. [00:15:00] Complicated protocol talked about on page 95. So many steps. Cannot test in one go. Too many steps, A through H. Even then one step had six elements in it, not very nice. [00:16:00] Continuing this work to separate the chains in various forms of derivatization. Beginning again with plasma, Saturday, October 21st, [ACD7B-39?] number 26370 is an acid citrate dextrose blood sample. [Outdated?] blood being used as a source of gamma globulin. [00:17:00] The new system with columns made up with several layers to prevent blockage of the column by settling. On page 117 basically a 4×20-centimeter column. Big columns. [00:18:00] Typical outline of procedure. Saturday, November 11th, page 127. Reducing with DTT 10 millimolar and then addition of higher concentration of [cysteamine?] dihydrochloride. And iodoacetamide, etc. Sunday stop the fractions. The collector had jammed. But the light chains are OK. Continuing these experiments. So Wednesday, [00:19:00] November 15th, page 131. Followed up on page 133 by 9:15 a.m. heavy almost out and by 1:00 p.m. light out. Light chains that is. Very good separation. Column problems.
Again the procedure Wednesday, November 22nd, gamma globulin to light plus heavy with the procedure written down quite clearly. [00:20:00] DTT, [cysteamine?] dihydrochloride, iodoacetamide, then urea and water. Make it acidic and run the gel. And run the column. Preparation of derivatives on Friday, December 1st. Decided to use [Dylan?] myeloma protein or light chain. That’s to say Bence-Jones protein. For making various derivatives. And the book ends on pages 152 and 153 [00:21:00] looking at the products of various procedures and formic acid gel with 8 molar urea. The gel was too fresh. The samples ran over. Repeat. So I reran on an old TVB gel. And said test all the products with a urea gel. [00:22:00] And so ends this book.