Oliver Smithies:[00:00:00] There is no transition between the planning notes of 1953 and the beginning of my experiments which began in December of 1953. So I don’t have any record specifically of what was going through my mind. But I do remember what was going through it. And that was the thought that the method of making insulin which was to use hydrochloric acid and diluted ethanol to extract material from the pancreas, that this method was very harsh [00:01:00] and likely to damage the proteins that were present. And so I thought that I would look for a precursor of insulin on the thought that this harsh method had resulted in some sort of degradation product. In fact there is as we now know a precursor of insulin, though as I’ve said in the past, I never found it. But that’s what I was beginning to look for in my first experiments at Connaught Labs under the supervision or the gentle guidance is perhaps a better word of David Scott.
So the first book that has any experiments is labeled [00:02:00] insulin. And it begins on as I said December 3rd, Thursday. And it’s talking about dissolving 45 milligrams of zinc insulin in bicarbonate buffer 10 ml and then doing various things to precipitate with acid alcohol. And the difference between doing this experiment in the cold and at room temperature. That in the cold when you added the acid alcohol there was an immediate precipitate. But if it was done at room temperature there was no precipitate. [00:03:00] And it goes on with that sort of experiment. I’m not sure what the aim was. But I called the final product cold neutral, CN, or warm neutral, WN, because the acid precipitates were neutralized with ammonium hydroxide.
Even though I’m starting with purified insulin makes it clear what some of the problems were. Dry precipitate was so small that could not get it off the paper. Expect preservation of about 60% of the activity, and so on. Large loss considering the amount of the precipitate. [00:04:00] Therefore try cold acid in cold followed by room temperature with filtration. And so we go. Trying again with alcohol added to bicarbonate solution insulin. So on. Similar experiments for a few days here. And a comment that D.A. Scott finds in original tests of 20 ml of alcohol added before standing at 5 degrees centigrade that this is more important to repeat first. I have no idea what that means.[00:05:00] December 8th I’m trying new zinc insulin lot 690. Just continuing doing experiments of this general type of precipitating insulin in different conditions starting with purified material. Then evidently thinking it might make a difference. New zinc-free insulin. Again lot 690, 120 milligrams of number 690 with a question mark whether it’s beef insulin or pork insulin. I wasn’t sure. And acetone and hydrochloric acid to get it into solution. [00:06:00] And precipitation. Working on Sunday I see here. Sunday, December 13th. That was a batch of solution opalescent pH 8.1 after solution. Zinc-free insulin. Nothing very striking at all. Yield about — here’s one where it changes from pencil to ink. Yield anyway 85%. Probably most loss in the first precipitation as addition of more acid to the supernatant caused more solid to appear. Could leave longer if yield is important, etc. [00:07:00] Quite a lot of pages covered in relatively few days here. Only a week later. Lot 901 of beef zinc insulin. Trying to understand the precipitation methods. Here’s a comment on December 16th. Only 56 milligrams of zinc-free insulin left. A new lot. I wasn’t nervous in using quite a quantity because I have a note here. [00:08:00] Zinc-free insulin from lot 901. Beef zinc insulin 5 grams at 150 mg per 4 ml. Must have been giving 5 grams of the material.
I was trying a big scale, 5 grams of insulin is an enormous amount of insulin. And here the next page saying that drying this large bulk not so satisfactory. Weighed yield 4.8 grams, 96%. I really did start with 5 grams of zinc insulin and tried precipitating it and got back 4.8 grams. And stored in a vacuum over concentrated sulfuric acid. [00:09:00] [00:10:00] Trying different reagents to precipitate the insulin. Note that apparently insulin hydrochloride is soluble in methyl alcohol and trying lauryl sulfate as a precipitating material. [00:11:00] Now in January. So there’s a fairly big gap there. January 4th is an entry and then the next entry is January 26th. Presumably I took some sort of vacation. Probably went to Wisconsin. Now I’m beginning to think about taking pancreas itself because on Tuesday, January 26th I start off with 20 grams of minced calf pancreas, [00:12:00] frozen calf pancreas. Cut up under the cold and in the presence of 0.25-molar sucrose, etc. So trying to replicate some of the previous methods of making it. And then all solutions were kept cold to this point. Which had been after the sucrose, there was homogenization. And the supernatant was taken off and centrifuged, decanted, and all the solutions were kept cold. To the supernatant I added alcohol. [00:13:00] And sulfuric acid. Concentrated sulfuric acid. So the acid alcohol method of making insulin. Note on what was happening. Three different procedures. Supernatant A was made 66% alcohol with two volumes of (inaudible) alcohol and sulfuric acid supernatant and various different fractions. [00:14:00] And the results were posted as standard over 30. I don’t know what that means. Standard over 30 gave — no, I really don’t know what it means. Though there’s A, B, C, D different fractions, 2/12, 12/12, 0/12, 0/12. This will be the number of mice that fell out of the assay cylinder. So the standard diluted 1/30 [00:15:00] 10 animals all fell out of the cage and with my solutions A, B, C, and D 2/12, 12/12, 0/12, 0/12. Though some activity is in the first homogenate. But most is in the unhomogenized tissue. Next step to improve the homogenization. So I’m learning how to make insulin really by pretty standard methods at this point. Or not learning, but trying to reproduce other people’s work. January 27th starting with 5 grams of pancreas chopped small while frozen, etc.
And then dividing into different fractions. And with the results shortly thereafter. The standard over 25 [00:16:00] and then my fractions which were A, B, C, D, 2/12, 10/12, 8/12, 12/12. With a comment this is absurd. And on checking the test boy, I find that the order really wasn’t that order. We made a mistake. Test boy, I don’t know what that means. There were some young kids evidently there in the assay room. I made good friends of at least two of them. Otto Hiller later became my technician. And [Jim Bryant?] was a good friend. And they both started off in the assay room having come to Canada as [00:17:00] immigrants under a program that would let them be hired by farmers. It was a sort of indentured labor. They could come to Canada if they worked with farmers for very low pay. But they all would try to escape the farms and get work, better work in Toronto. So these two guys had come as farmer slaves almost and had escaped to work in Connaught Labs and became my friends. Thursday starting with pancreas again, etc., etc. Pages of this sort.
Pages and pages of it. Trying sucrose and so on. [00:18:00] Beginning to think about electrophoresis. February 4th. Thursday, February 4th. Standard over 25, 12/24. A, B, C, and D fractions, 15/24, 5/24, 2/12, and 7/12. Thus much of the activity is brought down by centrifuging but a little, a very little, may be in the solution. [00:19:00] February 9th, Tuesday, February 9th. A comment that my solutions were sent to the test and evidently we thought about them as convulsion rather than just releasing from the rotating cylinder. Because of the remark underlined three times. No convulsions in any of the mouse. Must repeat with new solutions.[00:20:00] February 11th, all zero except for B. By now it’s got to be February 11th. And then there’s a big gap. And after Thursday or Friday, February 12th there’s a gap. And the next experiments are July 8th. Normal acid alcohol extractions used, 90% alcohol with 15 milliliters of concentrated HCl per liter. And I’m starting experiments again. The aim to precipitate insulin from alcohol extracts. Much the same topic that’s been bothering me all this time [00:21:00] up to this point. Not really very progressive. Not really very instructive. And then that ends this book with about a third of the book with just empty pages. Nothing in them at all. That’s my first book on insulin. Room 327 in the Connaught Medical Research Laboratories, College Street, Toronto, Ontario. Book one of insulin.