Oliver Smithies:[00:00:00] So this is the beginning of Book A, which was started on April, 1960 and this is the first entry after reaching Wisconsin from the University of Toronto. And it picks up on the same problem, or same task of making haptoglobin, starting with, again the pooling of blood, which proved in the end to be a mistake, with six bottles of plasma from the blood bank. And, as it happened, there were four, all four types of the haptoglobin that we knew at that time in these samples, 1-1 — well not all of them — [00:01:00] 1-1, 2-1 modified, as we called it, and 2-2. And the next few pages are just concerned with following the purification images of this starting plasma, and so on and so forth.
And already beginning to interact with the geneticist in Wisconsin because here, on page 5, April 5th is a sample from Klaus Patau, and Dr. Uchida, and Patau and Uchida, that had extra chromosomes, the father — and the mother and the baby of the one family, and the baby, mother and father of the other [00:02:00] family. Only one of the babies appears unusually in having a comparatively large amount of benzidine reacting material between free hemoglobin and HB11. I thought perhaps it might be fetal hemoglobin.
Repeating the samples again on April 6th, and staining with benzidine, and so on and so forth. I was looking at the precipitates that were material that appeared on the plasma preparation, the washed precipitates from [00:03:00] Tuesday, and the two unwashed ones for Wednesday were suspended in water, etc., etc. And, I looked at them. Not at this point, very clear what was being (inaudible). [Rather than being a true result?], HP2 appears to be present in the first and second unwashed [crop?] but not clearly in any other — etc., etc. on page 11.
More, looking at samples, comparing the supernatants with the whole, and different parts of the fractionation of the samples.[00:04:00] Thursday, April 7th, a rough pH 4.9 test and repeat, of the baby [whale?] sample, looking to purify the haptoglobin again. Bright yellow precipitate. Nothing very remarkable, just working at the purification.
April 8th and 9th. And continuing the next few days.
Beginning to do deionization on Monday April 11th, fusing the Dowex resins instead [00:05:00] of dialysis. And, absorption onto DEAE which is what I was using, or going to use anyway as the method of purification, a rough trial on page 25, 15mLs of the deionized pH 4.9-4.3 plasma, extracted with the DEA, and eluted with 0.15M sodium chloride, and the results show that clearly the haptoglobin is being recovered, though not completely pure, of course. Repeat on a larger scale, but acidify in one step, and deionize the media, that type of comment.
Continue with the purification [00:06:00] on April 1st, and — getting better fractions from the DEAE elution now, and in a larger scale, and saving the different fractions. Nothing very, very remarkable.
Wednesday April 13th, quite a nice gel showing that the purification is coming along well. Types [of?] plasma, from a bottle on April 13th.
Continuing with purification, getting large scale here, on April 14th, 2g of DEAE now. And [00:07:00] fraction — (laughter) — on page 37. They look bad in the note, that none were kept. Recovering the material on the 14th of April, continuing purification, nothing very remarkable.
More extensive test on April 18th, analysis (inaudible). Some samples to type, on April 18th, and so we go. Nothing at all remarkable, in any of the —
An interpretive [00:08:00] graph on April 19th, and on page 52, suggesting that all of the usual haptoglobin comes off as a certain concentration of — in certain fractions of the elution from DEAE, just documenting what was already shown in the gel. Continuing purifying material. Pages of the same general type, and diagrams of the elution pattern.
Higher pH tests [00:09:00] April 22nd, cold room tests. And so on, and so forth. Nothing very remarkable, or particularly exciting; just the old sort of experiments that have to be done to make a method reproducible, and obtain the product.
Trying a different adsorbent here, [ectiola?], which is a derivative of DEAE, with a comment on the DEAE [00:10:00] on April 29th. Some comments on the pH, but it was in a danger region for denaturing haptoglobin, but in the elution, but the protein had already eluted, but was not very significant.
Trying ectiola on page 79. And trying a Sephadex column. Slight fractionation, very [00:11:00] slight fractionation. Could be used as necessary, but better try chromatography with DEAE, and ectiola for the first, so just an attempt to get a separation by partition chromatography.
Page 85, rather or 84, a rather pretty diagram of pH, of the conductivity of the tip I used to follow where the salt came out, with lots of points, rather funny-shaped curves in the protein elution pattern to go with it.
Continuing ectiola experiments on page 87. [00:12:00] With a repeat here, of the comment of two ectiola experiment, “If anything worse than previous. Return to DEAE. If necessary, sharpen the gradient,” so ectiola didn’t look very promising.
And there, and in the following page is a fairly big experiment with DEAE cellulose, and careful following of the pH, and many, many points, and the conductivity, as judged by current in the tip that I used at the bottom of the column.
Trying an [00:13:00] ammonium acetate column on May 6th. Looked quite good. In fact, as judged by the gel on page 97, seeing essentially 100% hemoglobin-haptoglobin, and haptoglobin, [under line?], and there’s a rather nice gel result. And with a comment that, a very long column would even pull out hemoglobin-haptoglobin from haptoglobin, is a quite pleasing result on page 97.
So more tests of the same sort [00:14:00] with acetate and elution with ammonium acetate on page 99. The advantage I realized, I remember realizing of using the ammonium salts was that later on, we could get rid of them just by freeze-drying, or something of that sort, because ammonium acetate is basically a volatile salt. So here on Friday, May 6th, an acetate test again with fresh DEAE acetate, pH 4.6 etc., and eluting with ammonium acetate, instead of with sodium chloride.[00:15:00] But the result was not all that pleasing, (laughter), as commented on page 101, “Hemoglobin-haptoglobin separating from haptoglobin is much better, but not from albumin. The separation from albumin was very poor.”
So trying to improve on that, not doing very well on the following pages. 105, for example, shows albumin along with (inaudible). That’s page 105.
Calibration curves, nothing remarkable, just straightforward, trying to improve on the method.
Here we are on May — [00:16:00] more impurities, and no haptoglobin on page 119, and with comments on the recent preparation on page 123. Essentially, the same type of elution with distilled water and ammonium acetate and, etc., etc.
Rather pleased with the gel on page 127, no I guess not very pleased. It was just a check mark, but the result is not all that good. [00:17:00] As shown on page 129, “Results are again poor.” Test of a longer column, with a comment, “Very good,” and all sorts of graphs of pH and conductivity.
But, the gels on page 135 are disappointing. “Little separation.” 135.
More pages of the same type, and continuing, repeating the experiment with small variations. Noteworthy thing, like making a column with a [00:18:00] delayed section, due to Sephadex, etc. Nothing very remarkable, although I was evidently rather pleased on page 147, “Very good. The back is probably sharp. Try a gradient, etc.” And the haptoglobin is pretty pure on that, so that’s a rough test with Sephadex, delay section, starting on page 145, and ending on 147.
Continuing with this general idea, a long Sephadex test on page 155, and a graph showing where the hemoglobin-haptoglobin [00:19:00] came out, where the haptoglobin came out, and where the impurities came out, with a final comment on the last page of, “The results are essentially the same as 143, therefore abandon Sephadex delay and try lengthening the column, etc.,” and so ends book A.