Oliver Smithies:[00:00:00] Now, on book capital B, 1960, continuing the same slow progression of trying to get better purification. Thursday, June the 2nd is the first entry. And just, a rather unhappy-looking elution pattern on the following page. So made various columns, and pH 4.4 tests, et cetera. And nothing particularly surprising; pH 4.3 tests. And continuing in the same [00:01:00] vein.
The tests of pH 4.3 test, and on June 6th, here’s some fairly nice haptoglobin, as shown by the plot on page 13, and the gel on page 15, which was negative for benzidine. I mean, there wasn’t any — very much hemoglobin there. But again, nothing striking.
Continuing these, looks like variations of the same thing, but at least on page 21, I’m pleased, because I called this “Perfect columns and gradient,” pH 4.6 column and eluted under the same condition, [00:02:00] et cetera. One looks fairly good; one looks rather poor. pH 4.3 versus 4.6, makes an appreciable difference. So the 4.6 says, “Poor, yield no greater.” And impurities, some chromatography is needed to separate the haptoglobin from the impurities. pH is critical, 4.3-4.6 makes an appreciable difference. Nitty-gritty stuff. Nothing very striking.
Test with haptoglobin 1-1 now, which looked easier, June 9th, page 29, [00:03:00] where the results are very satisfactory. Results as usual, very good preparation. Trace slower impurity, but nothing greater than about 1%. This is [plasma?] #84, with haptoglobin 1-1 at pH 4.3 as documented on page 27, quite a pleasing-looking gel at this point.
Standard column with 13 gram on page 33, and the haptoglobin is fairly good in both of them, and the results show a standard column and a 1.5x standard column, and both very equivalent with quite good haptoglobin that looks like 2-1, probably. [00:04:00] More of the same, more of the same. More reproducible, evidently.
Second-stage tests, on page 15, June 14, with a comment on the following page that pH 4.3 is the best. pH 4.1 is promising, but pH 4.3 is the best.[Rust test?] pH 3.5, but [00:05:00] the result on page 55 doesn’t look very good. This was the result of that experiment which was not immediately clear. More tests, further pH tests in the low concentrations of ammonium acetate. Repeat, repeat, repeat, repeat. I’m always amazed at how many times I repeat these experiments trying to improve things, but I suppose that’s the way of my science, usually, trying to get things reproducible, and not be hesitant to repeat [00:06:00] things over and over again.
Here is the beginning of testing some of the purified solutions, on page 71, testing in the borate, urea borate gels. And more urea formate gels, which of course is the better way of doing it. Look at the separations now, a whole bunch of the materials made in the earlier pages, one, two, three, four, five, six, seven, or eight different pools of 2-1 haptoglobin separated most [00:07:00] usefully in the urea formate gels, where one can see the 2 band and the 1 band, and still this business of the individuals showing two bands in the 2-1, where the first band can be varied in position, and where when it’s a pool, there are three bands, still hasn’t — we still haven’t understood this. And haptoglobin pool of 1-1 showing two bands.
More pH tests. Microcolumns, a rough microcolumn setup. Micro-scale repeat, June 28th. Simplified procedure, et cetera. [00:08:00] Not too bad, steeper gradient tests on page 83, which is June 28th. A steeper gradient test will become very good, indistinguishable from normal, therefore adopt the simplified procedure, a simplified procedure.
Now back to understanding the degradation, or the deep polymerization, I should say, of haptoglobin, purified haptoglobin on page 85, June 29th, other reductance test, and iodine-free, iodoacetamide, because it’s some iodoacetamide preparations have some free iodine, [00:09:00] so it was better to purify it by recrystallization. I still have some bottles lying around of recrystallized iodoacetamide, and still worrying about this non-single zone of haptoglobin, possibly the non-single zone of HP1, and HP1-2, in the case of [B’9-0?] is due to incomplete S-S cleavage, so still think of it as a chemical problem, not having realized yet that it’s actually a genetic problem.
Here’s [00:10:00] Thursday June 30th, preparing 1-1 haptoglobin from a bottle of plasma. Trying with cleavage on haptoglobin stored in urea on June 30th, still thinking about chemical degradation.
A fast column test on July 1st, looked very nice from looking at it at this point of time. Further fast column tests on page 93, where there’s a good two-thirds, well a good half anyway, of haptoglobin is eluted pretty clean. More careful pH tests and so on. One got lost here — [00:11:00] no, a diagram got lost.
Extended pH test, et cetera. Here on Thursday, Wednesday-Thursday July 7th, attempting to crystallize 1-1. Recent preparations were greater than 85% pure, and possibly could be crystallized, and tried to see whether I could concentrate them down, and then scratch the tube et cetera. I’d rather it became cloudy and gave some stringy precipitate, but now, quite liquid, so don’t think I [00:12:00] was able to get crystallization. I was hoping it might work like haptoglobin.
More cleavage tests on page 107, with gels shown on page 109, but not very much comment on them.
Repeats of critical 1-1s and 2-2s on Sunday, July 10th. And [00:13:00] looking at the gels, with eight more urea and formic acid, at three different pHs, formic acid at 3, formic acid at pH 2.7, and citric acid at pH 2.4 to see if there was anything very different, that the gels are almost indistinguishable at this point.
On the following page, 3.6 citric, not much different. With a comment that pH 3.7 formic gel is very good; however continue the citric tests. But, it never was really adopted.
Control of reduction with mercaptoethanol [00:14:00] sodium sulfide, and so on, on July 15th. This is ambiguous; I think it’s talking about mercaptoethylamine, sodium sulfide, mercaptoacetic acid, and [sustain?], and mercaptoethanol, comparing all of them. The effective order of decreasing strength at this pH was that, it looked as if [00:15:00] — unclear. The [HC?] iodine test there to determine how well the material would reduce iodine to get some idea of the reducing power. Varied somewhat between samples, so mercaptoethylamine was 485 on this iodine test scale that I devised, and where mercaptoacetic was 275, with mercaptoethanol in the middle at 390. It’s unlikely to have made any real difference.
pH tests on reduction shown on July 15th, page 121 [00:16:00], 1-1 pHs ranging, from the reduction ranging from 3.4 to 6.4, and 1-1, 3.4 again to 6.2, and they’re pretty well indistinguishable. So, but no particular confusion stated, but all of them look to be the same. [00:17:00]
In view of the — Sunday July 17th, testing different concentrations of starch in the gels, with a comment that preliminary look suggests that 110% of the normal concentration of starch is the best. But, there was nothing terribly different about them.
More reduction tests going on, page 125, July 18. [00:18:00] Thinking of some sort of electrolytic test, with a lead electrode on page 127, July 18th. So here is a test with these different electrodes, rather obscure, not very well described, at least not by looking rather quickly. Reduction complete in less than 15 minutes, it says on page 129. [00:19:00] And trying to reduce electrolytically, apparently.[Consent?] about aging of samples in urea, again, Tuesday, July 19th, and so on and so forth. Again, this is a rather strange transition into [00:20:00] writing the different script, that’s not my writing. And the book ends with just confirming a lot of typing of different samples in the usual [multi-sort?] gel, [two?] gel, comparing many samples, for their haptoglobin genotype, and that ends book B.