Oliver Smithies:
[00:00:00] So now we’re on to the last book of the Roman numerals, Book XXIII, which begins on Sunday, January 3rd, and ends — I’ll look for the ending — in February of the same year, not a very long book. New fractionation tests, on January the 3rd. And continuation on Sunday of the 4‑molar urea, F‑20 test, with sodium lauryl sulfate. [00:01:00] And some tryptic digests. Thinking about fingerprints. George Connell — I mean Gordon Dixon was doing fingerprints and I got interested, for a little while, in doing some fingerprints myself. The comments are that “The tryptic digests of 1‑1, 2‑1, and 2‑2 fast components onto 3mls of Dowex 50.” And eluted with ammonium hydroxide and fingerprints made, and one-dimensional filter paper electrophoresis, in pyridine acetate. Etc. And so the fingerprints show a very marked [00:02:00] extra peptide in haptoglobin 1 but no loss in haptoglobin 2, etc. See over the page.Here gels looking at the fractionation of that — [oh, and?] the fast component. Some tests trying to get the treated haptoglobins to change their solubility, [00:03:00] with performic acid. And it’s suggested, “Only haptoglobin 1 is soluble, even to a slight extent, after oxidation,” so that the performic acid really wasn’t very helpful. (pause) Sunday, January 10th, “Freeze-dried haptoglobin 1, 2. Dissolved in dilute ammonium hydroxide. Not very soluble in water,” and, “Re-extracted with a larger [00:04:00] volume.” Testing oxidation. Copper — with sodium sul‑‑ looks like sodium sulfite and copper sulfate, with 5‑molar ammonium hydroxide, in solution. And so the gel shows that sodium boron hydride or Swan’s reagent, which is the copper one. And had no effect. But none of these reagents had any apparent effect on the haptoglobin, sodium boron hydride or [00:05:00] Swan’s reagent, copper sulfate — copper sulfide — no, copper sulfite. (pause) Trying to attach hemoglobin to haptoglobin again, without any success. Attempts to separate the three haptoglobin 1‑2 zones, January 11th, 12th. No indication of anything [00:06:00] remarkable. And dinitrofluorobenzene reaction, for molecular weight, by partial substitution, was [taking up?], Thursday, Friday, January 15th. But nothing was kept.
Trying to detect peptides in gels, ninhydrin, acetin, Folin’s reagent, Rydon’s reagent. “Promising.” “Exposed the gel to chlorine” and then allowed it to lose the free chloride, etc. Then potassium iodide. Following a paper of Rydon and Smith in [00:07:00] Nature, 1952. And found that only a few seconds of chlorine are necessary, that, as the gel dries, the chlorine penetrates — “Penetration drops rapidly” — and that it’s lost fairly quickly. Better procedure used later on. And a whole bunch of photographic images here, with no indication of where they are from or what they belong to, just loose photographs in the book. Some attempts to use bromethylamine, [00:08:00] for no obvious reasons. Made on Friday, February 12th. So haptoglobin 1, haptoglobin 2, bromethylamine, and iodoacetamide and iodoacetic acid. The aim is to check the three SH groups in [HV1?] versus two in HV2, by differential mobility changes. And the result says that it confirms that haptoglobin 1 has an extra SH group. [00:09:00] I’m not quite sure why that deduction was made. But I can imagine some of the reasons. (long pause)
Rather confusing experiment on Saturday, February 13th, with iodoacetic acid, [00:10:00] and para-aminosulfonic acid and sodium nitrite. So making a diazo salt, formed from sodium nitrite acting on para-aminosulfonic acid. And attempting to see what happened. And the result is that the diazo salts just messed everything up. All the diazo haptoglobins migrate to the positive, rather than the usual negative for reduced haptoglobin, [00:11:00] iodoacetamide in a formic acid gel. That’s… (pause) And Sunday, February 14th, sulfanilic, coupling repeated, with the same sort of result. Changes the mobility and everything runs the opposite direction. [00:12:00] Diazo experiment repeated, with [NH2-a benzene ring-CONHCH2COH?]. Gel run in reverse. But still essentially the same result as before. Attempt to get positive substitutions, on Tuesday, February 16th. (pause) Tests with [00:13:00] [Me2, and with pyridine?] — Me2 being, really — neither of them being pyridine, really. This was from Tuesday, February 16, with dimethylamino diazo compound — two diazo compounds, rather complex diazo compounds. I can’t anymore say their chemical names. But nothing very remarkable [00:14:00] resulted from their use, as the gel shows.
Began to consider the possibility that the haptoglobin preparation that gave the two bands with 1‑1 was due proteolysis. And so beginning to try DFP as a possible inhibitor of the proteolytic enzymes, in the preparation of haptoglobin. Yes, on Wednesday, February 17th, we began to investigate the possibility that proteolysis was responsible for getting two bands in the fast fraction of [00:15:00] haptoglobin 1‑1. So we began to think about using diisopropyl fluorophosphonate, a general inhibitor of the — of the proteolytic enzymes of general classes — the serine proteases, I g– And plasma was obtained from February 9th, a person, and purified. Etc., etc. And then the haptoglobin was made — [00:16:00] and, oh, was tested with and without hemoglobin, the purified material. That’s not very clear. Let me just try that again. (long pause) [00:17:00] And the material was fractionated in various ways and purified on diethylaminoethyl cellulose, and in the usual way, and to prepare the haptoglobin. With test of the material. With the material being tested on Thursday, February 18th, with and without hemoglobin. And [00:18:00] a trace of haptoglobin in fraction one. But fractions two to four contained good haptoglobin. And two small fast fractions. Estimated that I got 30 to 40 milligrams, total.
So, and goes on, and following days. Some purified haptoglobin from January 1960 and purified haptoglobin, type 2‑2, from October ’59 were tested. [00:19:00] There were two gels on Thursday, February 18th, one of which is really quire a nice gel. And the other looks a mess. It’s not clear what the difference is. But one of them shows clearly the ordinary type of result, haptoglobin 1 with two fast bands — of the [fast?] variety and haptoglobin 2 with a heavier band. This is complexing with the iodoacetic acid, instead of with iodoacetamide. So clearly, the [00:20:00] amide is much better. Perfectly straightforward and standard result on that gel. But alongside it is fraction number three. Very faintly, fraction number three from the gel shows — a small amount of number three, that “The plasma Hp 1 shows only one clear front band and possibly only one slow.” But the result here really is… I’ll backtrack on this description and go back to that again. So on Thursday, February 18th, we have the results of this experiment, where fraction number three, which is the haptoglobin made [00:21:00] after the DFP treatment… It’s only a small amount. But it shows only one band of the faster bands — lines up with the slow band of the haptoglobin 1‑1, amide, which shows the usual two bands, the haptoglobin being from January 1960, the purified one, and from — haptoglobin 2 from October ’59. So they gave the standard pattern. But the purified one shows only one clear front band and possibly only one slow. But haven’t yet realized what this means.
So Friday, February 19th. [00:22:00] Fractions three and four from the plasma haptoglobin — that’s to say, the DFP plasma haptoglobin — had concentrated down to a few mls, by concentrating them with Carbowax outside the dialysis tube. And 5mls was recovered, dried and from the frozen state. And I got 45 milligrams, about. And then treated in the usual way. And here the result is very clear, Friday, February 19, that the purified haptoglobin has only one band, the slower band of the two. “Results show multiple slow bands still present in [PHp?],” the [00:23:00] plasma haptoglobin, “but the faster looks essentially single, equivalent to the old slower zone.” So it was clear that this material contained only one of the slow components. This problem was eventually solved by George Connell, when he realized, by preparing samples from single batches of this sort, that there was a variation in the haptoglobin 1 gene. It could be 1F or 1S, as we later called them. But here was a clear example of this. Of course, this is being interpreted in terms of plasma and diisopropyl fluorophosphonate, not on one person versus a pool.
[00:24:00] Friday, January (sic) 19th, “Mixed-bed resin. Desalting of plasma,” etc. Plasma 2‑2 — haptoglobin 2‑2 obtained. The plasma 2‑2, I presume that means diethyl fluorophosphonate. (pause) No indication of the fluorophosphonate, though, in the — in the text. But purified haptoglobin is obtained, reasonably good haptoglobin, from the eluate, as shown on the — on the gel, [00:25:00] which follows, Friday, January 19th. (pause) Some problems with trying to acidify the 2‑2 haptoglobin. It looks like I lost the material. “Almost 100% lost of haptoglobin in the precipitate,” presumably haptoglobin fibrinogen, because of the use of plasma instead of serum. But I don’t know whether that’s correct or not.But Saturday, February [00:26:00] 20th, “In view of the haptoglobin loss in the precipitate, I took 5ml aliquots from a 1‑1 and 2‑2 plasma,” etc., and looked at the result. And the supernatants had haptoglobin OK. And the precipitates didn’t have haptoglobin. So, as it says, “Some haptoglobin in pH 4.6 material. Approximately zero in the rest of the 2‑2.” And the supernatant and the [00:27:00] precipitate, a trace of haptoglobin in the 4.6 and 4.31 fractions. But rather confusing results. (pause)
On Sunday, “60mls of 2‑2 acidified with Dowex 50 to pH 4.672 and dispensed into 10ml fractions and” diluted to various degrees with water and centrifuged. And put them on DEAE-cellulose. And only in one of the fractions is there appreciable haptoglobin. [00:28:00] Plasma is depleted of haptoglobin. So that was a bad idea. February 22nd, a mixed-bed resin acidification. No haptoglobin detected in the eluate. Etc. Problems. (pause) So this was pH — bulk treatment by making the… See how I made the pH.
[00:29:00] For Monday, February 22nd, decided to see if acidification with acetic acid precipitates the haptoglobin. Because I was getting trouble with mixed-bed resin change of pH — or acidic resin. And so I had pH 4.3 supernatant, 4.6, 4.9. And is Sephadex treatment material. And got precipitates which were comparable in amount to those [00:30:00] with the Dowex 50. But the result shows that, pH 4.9, the supernatant has essentially all of the haptoglobin. “pH 4.6, considerably less” and “pH 4.3, about zero haptoglobin.” So it’s easy to lose the haptoglobin, if the pH is not watched carefully.And Tuesday, February 23rd, is the beginning of almost the last experiment in these books. Alkaline DEA treatment. “50mls? of 2‑1 plasma desalted with free-base IR402 and free-acid IR120, [00:31:00] alternately, keeping the pH as close to 8 as possible,” in other words, one and then the other one and then the other. And got to pH 7.8, and [so?] quite high. And so it’s desalted with free-base and free-acid resins and then treated with DEAE-cellulose and with HCl, to pH 7, and washed and extracted with sodium chloride, and put on the test. The result that, “Some haptoglobin in #1 and #2 eluates but many other substances. And even [00:32:00] desalted plasma is low in haptoglobin.” So not very satisfactory result.
The last two entries in this book are acidification in the presence of DEAE, a couple of tests, a rough test and a better test. And a gel run. But no sign of haptoglobin, by color. “The reason for the trouble is aggregation of haptoglobin in acid solutions. See August 5th. Not preventable until 4‑molar urea is present. Therefore, use 4‑molar urea, by adding dilute acid containing 8‑molar urea, in the cold, to reduce denaturation.” And that’s the last entry in the Roman numeral books. [00:32:56]