Oliver Smithies:
This book, labeled 1953 Book I, physical I on the cover, is the start of my lab notebooks during the period that I worked with David Scott in Connaught Medical Research Laboratories. So it overlaps in part the book Insulin, labeled Insulin, which I recorded some comments [on] and clarify some of the uncertainties in that particular account. So here we go with 1953 Book I, the first experiments carried out in Toronto where I’m working in Room 327 of the School of Hygiene Building, College Street, Toronto. (I can only see one page at once. That’s not so good, in the end. I think I will turn it over with the book, I need to see both pages.)
The first experiment begins on October 27, and is my attempt to make the Macheboeuf method of filter paper electrophoresis suitable for my experiments with insulin. So I just used something that I could detect on filter paper, aniline phthalate and aniline phosphate in butanol, as prepared several months ago by Ted B., as indicators for paper electrophoresis with sugars, using number 54, Whatman paper, which I remember very well as a very hard paper that wouldn’t shed fibers. I liked that filter paper very much. And it starts off by saying “glucose spot more rapid than sucrose in developing color with aniline phthalate.” More intense. And I have some doubts about the quality of the aniline phthalate used in this experiment, because it had to be heated enough that the paper chars before you could see anything. Spraying was inconvenient and smelly, and so I later used dipping for uniformity. The aniline phthalate was dissolved in butanol, so on the page there were some tests of trying to detect the position of glucose or sucrose on filter paper using aniline phthalate. And then the next couple pages are similar experiments, just how long you heat it: 6 minutes, 9 minutes, 12 minutes, double dip, etc. etc. Glucose 2.5, blotted glucose, saturated, and so on. Not very satisfactory. Rather insensitive.
So now begin to try protein spots, and we have somewhere further down in the book insulin dissolved in about .1 NHCL using a ninhydrin of the stain in .5 molar acetic acid. It gives a fear (?) — you can see the fingerprints on the filter paper, but not insulin, which of course is because a ninhydrin reacts with free amino groups, with end terminal amino groups, not very sensitive with a protein. A ninhydrin gives clear fingerprint stains, but insulin spot is poorly colored. So not understanding how to detect protein on filter paper is the problem. And there’s a comment, which is the obvious solution, “look up wool or silk dyes, preferably black.” Realizing that I could use a wool dye instead of trying to do it chemically. I tried silver nitrate, ammonium hydroxide, blackness very rapid but too general. October, still setting up solutions in over 10 hydrochloric acid, not using dyes here. More experiments with filter paper, just not satisfactory at all. I don’t know why I’m not using the dyes yet, but I’m not. Many pages of filter paper strips, all very faint. And then here, on Monday, November 2, I got some azocarmine B solution from Poulik, I don’t even spell his name right, P-o-u-l-i-c-h instead of P-o-u-l-i-k, who later became a great friend of mine and a collaborator in many experiments. He worked for Fraser, who was the person who used to play golf with David Scott. Fraser had a lab down below on a different floor, and Dave Poulik worked there, and he had an azocarmine solution. I have a test here of the – the test gave color poorer than ninhydrin by several times. Retest with the same insulin and old concentration, similar and shows stain, etc. etc. easy to use and wash out but not yet working.
Evidently Gotz (?) began to get dye, a dye solution here, on Monday November 2, because I had a made a near solution of Amido S, which is Amido-Schwartz, Amido black, in 10% acetic acid and 50% methanol. I’m sure I got that recipe from Dave Poulik. Amido black became the stain which we used very extensively later on. So here is the beginning of Amido-Schwartz, as I then called it, compared with azocarmine B, and clearly the Amido-Schwartz was much more sensitive. Though I’m also aware of protein binding to the paper, because I have a comment here that “since absorption is important, full tests on thick washed paper were made, but don’t forget calcium paper might be better, so try this too.” I have something that I evidently thought was quite satisfactory, “repeated the experiment with more baths and a minimum of 10 minutes per bath, blot in between the baths. First bath removes the great majority of the unbound dye, so only this need be renewed each time.” So moving the filter paper from bath to bath to bath. I’m very happy with some sort of rocking apparatus that I made. I’ve got a big comment: “This is it!”
November 3: “Washing at 60 degrees gives very fast washing but makes the spots less defined and fainter.” Learning how to stain, learning how to stain. A comment here [to] “try stepping up the methanol if this doesn’t work. Tried 5 minute stain and if it works at 10 minutes it’s probably more than ample.” Also baking, with a comment that this was hopeless later on. So testing different dilutions now of insulin spots on the filter paper. Made from stock, .25% insulin in .1 HCl solutions, diluted in the following way. So a series of dilutions from .25% down to .0039%. And I could see the 1.5 micrograms. Baked at 110 degrees. Friday, November 6: began to set up the Macheboeuf drying method with filter paper and water and glucose on spots at various precisions and tried to see what would happen. So I have a comment here that after a good half an hour of evaporation, one strip was removed and tested and I could see where things had moved. Results very promising, and migration is definite. So I have an idea that the evaporation is moving the spots as was required to make Macheboeuf’s method work. Trying different temperatures. Rate very large with a comment that the spot moved so much in 20 minutes, and one spot was dry in the middle. Do not abandon these hard smooth texture papers. Still liking number 54.
And an experiment where the central line looks beautifully narrow, but cross evaporation is marked, evaporation moved the spots to the edge of the paper as well as along the paper. So I set up a wide 54 paper with central spots of ink and look to see what happens. Not happy with that, so I’m looking for water soluble color compound, especially as this butanol vapor is unpleasant. I can then use a dye, at least in the early tests, and I thought of using indigo as an excellent possibility as it should be harmless but its solubility is very low. I haven’t yet realized that this method is really not worth bothering with. Pages and pages of these tests. Monday, November 9, trouble with cross rod visible, cut it out, trouble, trouble, trouble. So I started to close the chamber and see what would happen. Results look a little better than the other paper but not much. And I have a note here: remember insulin is almost insoluble in water. November 11th, still trying it. They got so warm, some of these papers, that steaming occurred: “note that steaming occurs.”
It would appear that Saturday, November 14, was the last experiment in this book with the Macheboeuf method, at least there’s a change in heart, because Sunday, November 15, I began to — or Monday, November 16, I began to think about using electrophoresis, conventional electrophoresis. And I have a diagram of the circuit that I made, which contained a 30 milliamp meter in series with 4 resistors. Oddly enough, here in 2015, I still have that piece of apparatus, which was made 60 years earlier. That apparatus on this board. Circuit drawn (?) November 16. Worrying about electrolytic products in the electro chambers, and so set up a rather complicated system now using side strips. No, I haven’t quite given up Macheboeuf, I see. An interesting comment on Wednesday, November 18: the University DC supply failed at 5 pm and not on again for about 10 minutes. In those days, Toronto had still not made up its mind whether to use direct current or alternating current, at least in the University. So the University still had direct current supply voltages available in wall sockets. And the AC was, and I don’t suppose it still is, but in Toronto the AC was an alternating current at 50 cycles, instead of 60 cycles. And when you moved your hand across the — underneath a light illuminated by the 50 cycle, you could see the movement in little frames. The difference between the light retention at 1/60th of a second versus a 1/50th of a second. So Toronto still had 50 cycle AC and 200 volts, I think it was about 200 volts, DC. Quite dangerous. But it failed on that day, at 5 o’clock.
Still Macheboeufing. I really did think the method was worth using, but it really wasn’t. Sunday, November 22, I began to recognize a problem. Though the details are not clear, knowing what I later found out, it appears that the insulin is sticking to the filter paper, as there’s a comment here that “this is not nice.” And query: “lack of sodium ions?” I really was persistent, it’s something that really was a complete waste of time. Tuesday, November 24, set up with Gordon Dixon’s enzymes. Gordon Dixon was my friend, a graduate student working with Charlie Hanes. Results very faint, but about 2 cm migration towards (query) the anode. Still trying to do the air evaporation. And December 1, Tuesday, I set up 24 milligrams of insulin in 6 different positions and tried various things. Results not much different. They looked very poor. December 2, insulin moving on filter paper. Trying to do evaporation still. The results good except that the paper chars very easily. But confirmed that it is the paper used that is causing the trouble, as the bands are well defined here. Insulin and glucose. Sunday, December 6, no concern about what the day of the week is. Monday, December 7, trying to use now narrow papers, so that evaporation sideways is not a problem, so using very narrow strips of paper and with a comment that number 3 was a very narrow band with an exclamation mark. Although, my comment is that Monday, December 7, in the morning, the results are very poor. Must have had too little insulin on, so set up at 10:30 and took one off at 11:10, in this case I was more satisfied. The flow clearly is very considerable, so much so that on the next page I say “results: very sharp band. Extremely promising.” Pages and pages still on this wasted effort. Saturday, December 12, 12:30 pm, set up with a number 3 paper. December 14, query, siphoning taking place? Charlie Hanes, evidently, I’ve been talking to him about it, because Wednesday, December 16th I have a note that CSH suggested a glycerol, an anti-freeze, might cut down absorption.
Saturday, December 19th, Wednesday December 30th, Thursday, December 31st: here I am now trying to get insulin to migrate on a paper and I have sodium fluoride, sodium chloride, sodium bromide, and sodium acetate, and I’m beginning to realize that the insulin is just sticking to the paper and unrolling like a carpet. Trying to avoid the problem by trying different solutions as buffer. Sodium chloride, the main buffer component of the main electrolyte, with 1/100th (?) of normal zinc chloride, to have two positive ions, or sodium sulfate, to give two negative ions, or sodium nitrate, just to see if the iron made a difference. And looking at them, there really isn’t any appreciable improvement. All of them, insulin is absorbing to the paper and giving a schmear. Friday, January 1st, 1954, so I worked New Year’s Day, shows sodium chloride, sodium nitrate, sodium sulphate, and zinc chloride. And it’s a nice clear demonstration of the fact that the electrophoresis is in trouble because of absorption. I make the comment then: set up in .1 molal sodium chloride sulphate potassium ferricyanide to give a (???) and potassium ferrocyanide, but the ferrocyanide oxydized to the ferricyanide. Beginning to think that at the present stage, I may have to use a frontal type of paper for electrophoresis as none of these substances reduces the tail much as measured on longer times. Slowly getting to the point of realizing that I need to have a different material than filter paper.
Tried aluminum chloride, magnesium chloride, and glutamic acid. Note that on – This was Saturday, January 2, the aluminum chloride one charred at 150 degrees, try baking longer at 110. Monday, January 4, January 5. And here is the beginning of starch gel electrophoresis, the difference being on Thursday, January 7, I was using filter paper and having all these troubles, and then on January 23, I set up with dilute soluble starch, containing .1 molar H acetic acid and .01 molar magnesium chloride. And I made a slit in the jelly and introduced 1% insulin and ran, for ¾ of an hour, 220 volts; that’s the DC voltage in the University DC power sockets. Results found very promising.
So here I’ll pause from the account of the journal and – no, it’s still recording? – I’ll pause from the journal and explain what was going on, which is not recorded here. And that is that on that Saturday morning, I heard that in the local hospital for sick children they were using the Kunkel and Slater starch grain method of electrophoresis, which I’d noticed in my planning notes. This method consisted of making a tray, probably about 12 inches long, maybe about 6 inches wide, and ½ an inch deep, and filling the tray with starch grains, starch grains, not starch solution or anything. And then put in buffer, moistening the whole system with buffer, so the proteins now migrating around the starch grains in the buffer, which is moistening this whole box. Rather like a wet sandbox, except it’s a wet starch grain box. And Kunkel and Slater had shown this method, that proteins did not stick to the starch, although they knew very well that proteins stuck to filter paper. So this was their improvement. However the downside of it was that in order to find the protein, you had to cut the moist starch grain block, as it were, into many slices and do a protein determination on every slide. So although the results were very pretty, the labor was enormous because you’d have to do 40 protein determinations to get one run out of the system.
And I saw this on that morning, in 1954. And then I remembered helping my mother — and I always put this in quotation marks, “helping my mother”, at least I was there when she was doing the laundry — and in tidying up, noticed that she’d made a starch, a soluble starch gooey material to allow starching the colors of my father’s shirts. And when you tied it up, this would be jelly, I found, I noticed, and evidently remembered because I thought, well, let me take the starch and instead of trying to make a moist bed, I’d cook the starch and make a jelly, and then I could stain the jelly with Amido black, or whatever, and see the pattern with very little work, relative to 40 protein determinations. And so I went back to Connaught Labs and went around various stock rooms, because Saturday morning, nobody around, and I had a key that could get me into any place, and I knew the stock room. So I found some starch powder which was called Starch According to Lintner, I didn’t know what that meant at that time, but I found that if you took this starch you could heat it up in water or in a buffer and it would dissolve or become soluble and, if allowed to set, would make a jelly at a high concentration. So I went back and set up dilute soluble starch jelly, containing .1 normal HCL and .01 normal magnesium chloride, with a question mark about whether that was necessary, and made a slit in the jelly, introduced the insulin into it, and ran it for ¾ of an hour at 220 volts, and the result was very promising when I stained the gel, although only surface staining was possible. But the insulin migrated as a beautiful band, not a bit of schmearing. S that was the beginning of starch gel electrophoresis. Very rapidly thereafter, I began to realize that this was a useful method. I didn’t do any more experiments with filter paper at all. Here I am Sunday, January 26th, set up at 11:50 at 110 volts, etc. Different slits, different little problems. Tuesday, January, set up with a good slit, and began to understand that you had to look at a slice of the gel, rather than at the surface, because of some distortion during the migration. Keeping going. Tuesday, December 1st, zinc free insulin again.
And the end of this book refers to the insulin work, December 1st with Dr. David A. Scott, beginning to do various assays in the insulin work, and that’s why the Insulin book is in a sense in parallel with this that I called physical I and Insulin are two books that are running in parallel. Just one entry in the physical book referring to December 1st, with David A. Scott, with D.A. Scott. They’re trying to precipitate insulin, and that’s what we saw in the Insulin book, that the Insulin book begins that same date, December 2nd. In the physical notebook, Tuesday, February 2, is the beginning of some of these experiments. I’m a little confused there. And Tuesday, December 3, in one book, and Wednesday, December 2, in the other book. So there is the end of one book and the beginning of another book, consecutive. So the Insulin book is consecutive to, it follows 1953 (I), then Insulin, the book, is really the next one in the series. And we’ll look and see where physical II occurs. [unintelligible]