Oliver Smithies:
So book Roman numeral XII starts April 1957, and runs through July of the same year, and starts again with the haptoglobin purification continued. So acetic acid wash [DOWEX?], allowed to settle, etc., serum added, divided into three parts, and one was distilled water — oh, added hemoglobin to each, and then divided into three parts, distilled water, 0.01 molar, sodium chloride 0.05 molar, and looking at the gels. It came out [00:01:00] the haptoglobin increases approximately linearly with an increase in ionic strength. In fact, so eluting with 0.05 molar sodium chloride, and hemoglobin gave even more. So I increased the ionic strength of 0.05 molar and made a comment to try even more.
So Tuesday, April 16th, continuing the [argument?], and the results show very little increase in haptoglobin with increased ionic strength. So, for example, 0.2 molar shows two [fast?] impurities; [00:02:00] [1?] molar shows a trace of the fast; 0.05 molar shows minute trace of impurity, but 0.05 molar… It’s not clear what that means, 0.05, 0.05 shows no haptoglobin at all, and no impurities. So what that meant, I’m not sure. Not very important to worry about. I’m beginning to think I might as well do this with a column rather than with powders, so I made a crude column test, and I put the resin above some starch powder in a little column, and I ran through 0.05 mils of serum, and then water, and then eluted with [00:03:00] 3% hemoglobin [in?] gravity, and (laughs) comment, neither any good.
Oh, April 16, 17, and I’m trying to see if I could get any copper binding protein, and a millicurie of CU64 was injected at 8:00 p.m. into wherever. I think probably this must have been into a [00:04:00] sample of serum, with… And I had — oh no, this is probably what had been used. These were sampled from a person with Wilson’s disease, and the clinical tests were still — used radioactive copper 64 with a patient who was thought to have Wilson’s disease, and I was given these samples to look at from this individual. So the injection was at 8:00 p.m., and then samples were obtained at 8:15 p.m., 9:30, 11:05 p.m., and 2:15 a.m. Then I got these samples [00:05:00] and ran them on the gel, and were put on to film. So I tried to do an autoradiograph of them. Put it over two sheets of x-ray film at 10:25 p.m., all having run the gel. Used two small strips, soaked filter paper, as marked, and the film was put on top. And I don’t know what the result is. It’ll no doubt appear. [00:06:00] I don’t see the results of that experiment yet. Maybe it’ll turn up later in the book.
And back to haptoglobin purification, with 10 mils of fresh DOWEX, etc., and got some binding material, haptoglobin OK, it says. Thursday, April 18th, continuing there with DOWEX 50, sodium salt, and there’s a little sulfonic acid — that must’ve been sulfuric acid, I’m sure — [00:07:00] to bring pH to 5.2, and as of the hemoglobin, to get it to bind, etc., and did the usual trick of eluting with 0.3% hemoglobin in distilled water. Negative results for haptoglobin, so that 0.3% hemoglobin in distilled water didn’t elute anything. Still a little puzzled, so DOWEX — in the following page, DOWEX 50 and DOWEX 2 were tested, all negative, not kept. April 18, 19th, [00:08:00] it was an attempt to do something with glass beads. It’s not at all clear what this was about, to see if I could get the hemoglobin to bind to glass beads, I presume, because they were — glass beads were soaked in hot potassium hydroxide, and then concentrated HCl, and washed. pH in the acid was 5.3, and the alkaline was 8.4, and added hemoglobin to the suspensions, etc., trying to use glass beads instead of the DOWEX resins, all completely negative for haptoglobin, so glass beads didn’t work.
Monday, April 22nd, more tests with DOWEX 2 [00:09:00] acetate. Both are negative, the two tests. Continuing with this on some column tests, which were various fractions collected, but let me go through that again, because this is a critical experiment that told me what was happening. So I go over this [00:10:00] experiment again. On Monday, April 22nd, 20 mils of packed DOWEX acetate were washed, and 5 mil small samples were made up to 50% with distilled water, and as a freshly thawed serum to the larger batch, and a smaller amount of older serum to the other batch, and washed four times with distilled water, each case, and extracted those small portion for tests, [00:11:00] with a comment that both the tests were negative. These first experiments were not kept — these first experiments do not pay, is the comment, to check if hemoglobin extraction is the cause, extracted a second portion with 0.05 sodium chloride hemoglobin, an OK result. So a comment on the left-hand page. This was evidently the column that was — [I made a?] different column. Must repeat this Thursday, Friday experiments with sodium chloride hemoglobin. So repeat on Thursday was plasma, ALO plasma in the usual way, [00:12:00] and eluted with 0.05 sodium chloride and hemoglobin, and then MJ plasma in the usual way, eluted with 0.05 molar sodium chloride and hemoglobin, and a control, a 0.05 molar sodium chloride, and the result is that it showed that hemoglobin was not necessary, that I got plenty of sample of haptoglobin off with MJ sample with just sodium chloride, not quite as much as I got with the sodium chloride hemoglobin, but it showed that hemoglobin was not necessary to elute the sample. I have a column test with various fractions from the column, [00:13:00] but with some problems of the filter paper absorption in the electrophoresis, but the various fractions from this column test showed that you’ve got a lot of haptoglobin that came off in fraction two. Thus, haptoglobin comes off with the earliest [front?], and can probably, therefore, be got off at a lower ionic strength, especially if the pH is kept low during absorption. And a big comment: hemoglobin is unnecessary. So hemoglobin is not necessary to elute the material from the DOWEX resin, and the DOWEX resin didn’t appear to be red [00:14:00] when hemoglobin was run down initially, suggesting that hemoglobin had nothing to do with the whole system.
A glass bead test on April 23rd, both negative, not kept. Glass no good for [straight work?]. Repeat of the [acetic acid?] bonding experiment with DOWEX 2 [acetate?], and MJ serum washed with distilled water, and the pH of the third wash was 5.1, and extracted with 0.01 molar sodium chloride, and no hemoglobin at all in this experiment. One experiment, and the other [00:15:00] experiment was washed with — in attempt to extract, etc., etc. And so it was a result of this experiment that almost approximately zero of pH 5.1, where the usual method gave haptoglobin in the usual way, whereas the pH was 3.4, and so approximately zero is the comment, with a CD assay binding, so therefore the isoelectric point of haptoglobin is probably greater than 3.5. I’m getting there, not quite there yet.
[00:16:00] A comment that on the next page I tested samples in formate buffer, acetate buffer, and the usual borate buffer, and looking for the migration of a haptoglobin seems likely that the isoelectric point is in the region of 4.6 to 4.8. It is possible that accurate pH can have around 4.7, would elute the fast impurity, but leave the haptoglobin on the resin. So I am realizing I can use this to purify the haptoglobin. A comment, still doing it in columns, which are a little bit more clumsy, but allow a larger amount. [00:17:00] Following the elution with conductivity measurements of when the sodium chloride comes out. April 24, 4.5 mils of freshly [so altahila?] serum, in 90 mils total volume of distilled water, plus the resin washed in 2 liters of water and decanted, etc., etc., made into a column. So I did the binding in solution and then put it into a column to elute it, and commenced — so no hemoglobin at all in this [00:18:00] experiment up to this point, and commenced washing it with 0.05 molar sodium chloride, and watching the pH — and watching the conductivity and the frothing of the material that it came up in different fractions. So eluting, starting at 3:55 p.m., with 0.5 molars sodium chlorite, and getting the resistance, and also the time, and so that began at 3:55, and at 5:15, by then the conductivity had begun to increase [of the early rate?], and went down to — quite rapidly — to a pH at that time 5.5. [00:19:00] At 5:21 p.m. the resistance was now quite tenfold lower than it started, and it was frothing, suggesting there was protein in the 5:21 p.m. sample, which we should see the answer shortly, with an interlude of a family study. The first 15 samples were tested on April 24, and the results showed that faction one had — let’s see what… Oh, yes. Choose one to ten. About 0.4 milliliters per tube, 11 to 15, about 0.8 milliliters, etc. And so the first 15 test samples were run on the gel, [00:20:00] after being eluted with 0.05 molar sodium chloride, and the results are sample number four. Two and three and four had haptoglobin, which three was very strong. So the comments are the following: the detection system is quite safe, and a drop of resistance of 50% is as equipped to tell me when to start collecting things, the fast impurities, the [more shovel?], but haptoglobin comes off so readily that probably 0.02 molar sodium chloride is adequate. So it’s now refining the system. And so it is that you don’t need hemoglobin [mount?] of the column, and you don’t need hemoglobin [00:21:00] to elute from the column. It’s all a matter of ionic absorption at the suitable pH in elution with sodium chloride. So on this gel, I have a nice result sketched at the usual gel fast impurity probably argument, and sure could be removed by a second absorption. And the acetate shows the result confirms previous work, that the isoelectric point is close to pH 4.7. So it could first try dilute acetic acid extraction, etc., trying to refine things.Doing this at April 25th, Thursday, so now I’m refining the system, but I obviously [00:22:00] have a way of preparing haptoglobin. Turned out to be very reliable and very secure method of getting haptoglobin, quite relatively pure from serum in a single-step column. Now, the family study tucked in there, April 26th, bigger samples. And some comment here that trying acetic acid as an eluent, but acetic acid does not displace the haptoglobin, but might be used to remove the fast impurity, especially after a second absorption. Must try lower sodium chloride on the column, etc. [00:23:00]
And Saturday, April 27th, and different detection system, a column, etc., and elution. And the results of elution with 0.02 more sodium chloride and haptoglobin is still obtained in fractions two and three. I’m beginning, I think, to collect the fractions when the conductivity has begun to change. And more columns, more tests, [00:24:00] April 29th, Monday, April 29th, pH, etc. Results were expect haptoglobin in fractions two and three. The results show that number two is a maximum, but there’s still some as long afterwards as five, and I’m worrying now about the column being a good column. Continuing of the same vein.
Some comment now written by Otto. Maybe he was writing down and I was doing it. I don’t know. [00:25:00] May 1st, tried DEAE cellulose instead of DOWEX. Not kept. I must’ve had some sort of reference here of the use of DEAE cellulose, because I have a comment here that the original reference suggests that alpha-2 cannot be got off at either end of a chromatogram, so stick to DOWEX. [00:26:00]
Thursday, May 9th, similar experiments to try to control the pH of the serum to a level where most of the impurities do not go on to the resin, because looking back at an experiment with resin hydrochloride on April 15th where no fast [00:27:00] impurities were present, so I take the usual 10% dialyzed serum, which is pH 6.65, and adjust the pH with acetic acid, 4, 4.5, and 5. Getting some clear precipitate, not surprising, in columns overnight. I’m quite puzzled by the fact that quite a lot of this writing is not my writing, and yet I’m describing experiments that I’m doing, so it’s possible I was dictating this, these writings. It’s a little bit strange. [00:28:00] Tried DOWEX 1, the comment that as here used it’s unsuitable. Having some difficulty reproducing results, because — there’s a note that May 7th — this is a note written on probably May 15th or 14th, note that May 7th was the last result with a high haptoglobin, and this was the last with four mils of serum, etc. Didn’t agree with that difficulty. And Otto — well, maybe there are two experiments going on at once, because here’s a comment [00:29:00] outlined very heavily with markings — Otto Hiller has been using two to three minutes, and I have been using 15 minutes. In elution. Let’s stop.
More pages of material in Otto’s writing. A comment on May 21st that this experiment was designed to see if the rather low concentrations of haptoglobin in recent experiments [00:30:00] were due to [set me?]. Careful check at all stages was made, but still the concentration rather low. Must try different things, etc. And that’s where I’ll stop today. I’m a little tired.