My book, physical three, continues the story that ended in book two — or that was present in book two, of worrying about various components, what things were real, and what weren’t real, and why the albumin spread out, etc., etc. And so to Thursday, April 29th, is the beginning, and just trying different ways of inserting the sample into the gel. [00:01:00] One was with reconstituted cellulose, etc., and nothing very striking. And indeed, the next page says that the results are highly suspicious, though the intensity of prealbumin in one suggests an effective quantity of plasma use.[00:02:00]
I am worried about whether the prealbumins are real or not. And attempting to isolate them and purify them, or show that they can be taken out of a gel and re– and run again, and shown to be present.[00:03:00]
On Friday, April 30th, I began to try to get the prealbumins cup from the gel. So I have a note that the sample was set up with reconstituted cellulose on Thursday, April 29th. And then on Friday, April 30th, I sliced the gel to try to get good material out of it. [00:04:00] Part of the gel was stained, and then part of it was used for preparing the prealbumins. I say, one and two were stained, that’s where slices one and two from the outside of the gel, and then two slices from within the gel, three and four.
So I commented the whole gel was then marked with a multiblade cutter, and number three gel was stained. But I now know where the samples are from. Even by [00:05:00] looking at the stain [frag part?], and comparing it with the unstained part. And so cut out section 12 and forwards. And I should say, the albumin band had a front at 11, and so sections 12 and forward contain the prealbumins, and were taken out and frozen. And then I stained the gel afterwards to see if the cup was good, and I say that I stained rest of four, a very good cut, should contain all of the prealbumins, one and two, without any albumin. And the cut was safe, as at no place does albumin cross the line. So I now have prealbumins [00:06:00] separated from albumin.
And the result is then tested a few pages later. At 4:20 p.m., I set up the prealbumins again, dissolved in distilled water, and compared them alongside [my?] plasma. And with no possibility of anything flowing across the gel from the plasma to the place where the prealbumins were, on the photograph, the sketch is [00:07:00] very faint. But nonetheless, it says the results show prealbumin is real. So I no longer am worried about prealbumin being real.
Looking at the effect on Saturday, May the 1st, of loading the samples with the pre-washed, starched grains, versus unwashed, and the differences are very small.[00:08:00]
Another experiment with the prealbumins, Monday, May the 3rd, with a comment that the great distance of migration of the prealbumins in a different gel suggests that they are lower molecular weight, or perhaps strongly acidic. But again, I compare the prealbumins with plasma, and they could see certainly the faster of the two clearly, and the second one just about. But again, no question that the prealbumins are real. [00:09:00]
I’m worrying about the plasma containing hemoglobin, and on Friday, May the 7th, I commented that the blood was taken into a cold, 40-milliliter, siliconized tube, [centrifuge?] 10 minutes at 4,000 in the cold, [00:10:00] and then eight milliliters withdrawn. Potassium fluoride and potassium oxides were added, to give 0.1% of each, as is used at the sick kids, meaning that’s what they used in the sick children’s hospital in their sample.
Here’s an example of how the work was going, which is certainly entertaining, that on Monday, May the 10th, 10 milliliters of blood [00:11:00] plus half of a mill of 0.1 molar sodium oxalate was centrifuged at 6,000, supernatant removed and centrifuged, etc., to prepare the material.
But the 10-milliliter blood was allowed with the — I don’t understand this one. Backtrack to that one. On Monday, May the 10th, it didn’t clot. I know what the point is, but I can’t really see it here. This was [00:12:00] blood was taken into a centrifuge tube, which had been coated on the inside with Vaseline, by taking Vaseline and heating it to a good high temperature, and putting it into a tube, a 50-milliliter tube, letting it all get very hot, and then inverting it so that it drained, left a thin coating of Vaseline on the inside of the tube. That was therefore no water — no aquaphilic surface was presented to the blood. And 10 milliliters of blood were taken without adding anything. And up to 10 minutes, 6,000 RPM, [00:13:00] the supernatant was taken off. And the other 10 milliliter was allowed to clot. You know, so here were two samples, 10 milliliters taken into oxalate, and 10 milliliters taken into the tube without oxalate. And then note here that although that was taken at some time in the morning, not cluttered by 6:00 p.m., so the Vaseline tubes are very effective. And in fact, I found that I had to add a little bit of filter paper to get those samples to clot, they were so resistant to clotting at that point because there’s nothing to stop the clotting system.
But a very good way of preparing plasma so that the clot would retract properly [00:14:00] from the vessel wall without tearing the red cell, and you could get hemoglobin-free serum in that way. But you had to seed the clotting system by adding a little bit of filter paper. I used it a great deal afterwards, that method.
A sample was tried with purified albumin, looking for where the albumin was taken from. Yes, it was prepared on Monday, May the 10th, [00:15:00] at the time in which the prealbumins were taken, a little bit of the albumin part of the gel was used to prepare albumin with a note that it will probably contain alpha one-two. I don’t know why that note is there, but it was a concern. But that sample was then tested on the following day, and compared to albumin serum plus fluoride and plasma. And the result was that you could see that albumin indeed ran as expected alongside the plasma.[16:00]
A test made on Friday, May the 14th of three gels, being at 7.1, 7.9, and 8.3, in which I can see that the sharpness of the albumin band varies with B8. So 7.1, it’s sharp at the back, and at 8.3, it’s sharp at the front, [00:17:00] and at 7.9, it was about equal front and back. So the sharpness of the albumin is partly dependent upon the pH. And as we later see, it’s very understandable but annoying.
The next page is Friday, May 14th, looking at pH 8.32, 8.68, and 8.98. And that they — the sharpness of the albumin is sharp at the back of 8.68 and sharp at the front of 8.98. Oddly enough, 8.32 looks to be about equal. Not clear why, in that case.[00:18:00]
A similar attempt was made to convince my [cell phone?] successfully that these components are real and not artifacts was to try to purify the [faint binds?] which appeared behind albumin in the gel, which I called alpha subscript 1-1. So I prepared, you know, S&F plus albumin, and also tried to get the slower [fraction?]. [00:19:00] And as a result, I’m showing that one can purify the components that run behind albumin, so that they’re also real. So I was obviously worried about the possibility that the [bandwidth?] I’ve been seeing were artifacts and not real. But everything said that they were real, although the sharpness of the band in the [electrofluency?] depended upon the conditions of pH and concentration of the buffers.
Here’s on Thursday, May the 20th, an experiment in which the alpha 1-1 and the F were prepared [00:20:00] and should be pure, and then were tested again against plasma, or serum. So an experiment where only the slower component of the prealbumins, the slow versus the fast of the prealbumins was prepared. And as expected, the purified material lines up with that that’s present in plasma, or serum.
Worrying again about hemoglobin, repeat the serum results with increasing amount of hemoglobin. And [00:21:00] then with serum versus plasma. And another test that says again that the fraction S-F from yesterday, which is — this is Sunday, May 23rd — S-F prepared from yesterday, versus a new fraction, very gooey. The starch was very gooey. But nonetheless, it confirms that S and F are real by this technique.[00:22:00]
(laughter) A note here. It’s not obvious what this means, but it’s a sketch of a gel, and it says, “Poor, not kept.” I should have commented at some stage that all of these stained gels were kept in test tubes full of the de-staining buffer, with a trace of dye left in them so that they didn’t wash out completely, and kept sealed. And some of these, I have a couple of these still left from 50 years ago [00:23:00] in tubes sealed with a rubber stopper. And they’re still — the gel is perfectly stable, and you can still see all of the bands that are there, and they — they kept indefinitely, if looked after. And I kept all of them until I left Toronto, and then I think I threw away a high proportion of them, because they really weren’t needed. But on June 2nd, I show a method of making [averse light?] in the gel better with a little tray that’s diagramed in the left-hand page.[00:24:00]
Still wondering about fibrinogen. Not really convinced that I can see fibrinogen in the gel. My later knowledge suggests that fibrinogen [00:25:00] never entered the gel, that it was too big to enter gels. But some proteins are so big that they don’t even enter a gel if it’s concentrated. It’s not that they migrate very slowly in the gel. They never enter the gel.
Evidently, every now and then, I would get an experiment that I liked, because setting up some experiment on Wednesday, June 9th, I have a comment that they — that [00:26:00] on — that the test with serum, that two pHs, 8.68 and 8.98. And the next page says, “Both beautifully set up with wet papers,” 7.98 is one pH, and 8.32 plus or minus 0.02. So good that the [slerums?] are visible at 20 minutes. And second [slerums?] about 0.5 millimeters out, also probably beta.
And on the left-hand side of the page are two sketches which underline doubly, “Very good.” I must have been pleased with that day. Next day, not so good with Thursday, the sketch on the left says “Moderate” with one underline [00:27:00] instead of “Very good” with two underlines. (laughter) And following page, I’m pleased again, “Very good,” but only one underline, 7.90 and 8.25, the gel.
An experiment here were I got a result that I wasn’t very happy with. On Tuesday, July the 6th, and have something odd about the experiment, though the gel [00:28:00] wasn’t kept. And there’s something variable here. And on the following page, I say I suspect the starch percentage most. So I checked a typical transfer on an accurate balance, and found that they got 14.7 instead of 15 grams. So in the future, weigh accurately, and pipe at 100 milliliters to make sure that the precision was correct. Always worrying about precisions.[00:29:00]
Some general observation made on Wednesday, July the 14th. “General observation: it’s now pretty clear that around four hours is necessary for full resolution of globulins.” And I think later on, I settled on running gels for four hours. Although that can’t be quite right, because I remember somebody commenting that my gels ran eight hours, and they couldn’t — so later on, I must have changed to eight hours, because some friends in England say that, well, we knew you were a bachelor because you were running your gels for eight hours. And you can’t do an experiment of eight hours in the normal working day. (laughter) There isn’t enough time to [00:30:00] make the gel, set it up, run it, and then take the results up. So you must have been working at times that went more than eight hours. And they deduced I was a bachelor. (laughter) They were right.
And that one that says general observation for around four hours. I see in looking more carefully that there was three underneath it, and then four was [overwritten in?] rather heavily. And so in fact, it ended up by being eight hours, not four.
More and more little details, trying to get things reproducible, understandable. I still am never frightened to repeat an experiment many times. I’m never happy with [00:31:00] an experiment that hasn’t been repeated at least three times. I mean, let’s just say it has been done three times, at least two repeat. Perhaps sometimes, if I get the same result twice, I might accept it. But usually, like more.
I added some insulin to a gel, [rabbit serum?], plus considerable hemoglobin. And I added 0.1% of solid insulin to it and ran it, and found that the insulin in fact ran faster than albumin. Insulin shows, with an exclamation mark, in the position where the prealbumins were. But the rabbit serum didn’t have any prealbumins. So note, no prealbumins. Repeat on [00:32:00] human to see what I could see on him. Whether I did it again, I don’t know. We’ll find out. Here, human serum, and serum plus insulin. Yes, here it is.
Gel not kept, though. Couple more gels here with the comment, “Not kept, not kept.” Not many of those. I kept most of them. [00:33:00]
Trying gels at different temperatures in the cold and at room temperature, and just differences in migration, distance, but not really in results. Almost indistinguishable is the comment of cold versus room temperature.[00:34:00] I’m beginning to — beginning to worry about something that’s making the albumin get sharp, at the front or the back. And I’m thinking, has it something to do with the voltage gradient? So I made a little rack with electrodes, copper electrodes, at every three or four millimeters. [However?], in this one initially, every nine millimeters. So a piece of plastic with every nine millimeters, a little copper piece of wire that’s stuck in. And I used these and made a lot of measurements. And I still have one of them left, and all the copper electrodes have obviously also electrolyzed during the experiment, because they’ve become tapered. But I use them to measure voltage [00:35:00] gradient along the gel to start to understand what was happening.
And my first experiment was — maybe it was not made with a grid, but just by hand. Roughly every nine meters, a voltage gradient. And I can see these voltages are measured at 6.2, 6.2, 6.2. And then one is 17.2, and then 7, 8, 8.1, etc., etc. So there’s obviously a part of the gel where the voltage gradient is very different. And I imagine it’s the front of the albumin there. And we’ll see. No, this would likely be the back of the albumin, so that the voltage gradient is very high. And so if anything, it drops behind, it speeds up so that [00:36:00] this is going to explain the shape of the albumin band. But I haven’t yet reached that conclusion.
And another pH, another voltage per centimeter. Quite variable along the gel. 5.6, 7.2, 7.6, etc., etc. Up 30 minutes, up all of these measurements. Where the protein region is a voltage [gradiented?]. Instead of being about 7.0, 6.9, 7.3 volts, it’s dropped down to 8.2 and 5.8 in the front, and behind it is 8.0. So I’m beginning to realize that the voltage per centimeter is quite variable [00:37:00] along the gel even at 30 minutes. And at 60 minutes, the variation can also be seen up and down.
And the more and more examples of my friend, the voltage gradient, to understand what was happening. And have one place where it says — where there is protein. Where the voltage gradient is fairly constant, but where the [00:38:00] back of the protein is where the voltage gradient is different, the 7.8 at the back. So it’s not due to the protein itself, but the voltage gradient near the protein.
Wednesday, August 25th, a mammoth experiment here, with many, many measurements of voltage gradient along the gel. And on the left-hand side, what the voltage gradient looked like with the two gels, one of which was [borate?] with thousandth-molar sodium chloride, about 0.04 molar borate, and thousandth molar sodium chloride, and one with sodium borate, [00:39:00] 0.04 molar, and five ten thousandths sodium chloride. And measuring the voltage gradient, and the sketch of the protein. And it’s clear that the gradient is behind the protein in a rather strange pattern, a sigmoidal pattern. Above normal behind, and below normal, ahead of the protein, with a comment, “Very good.” I like that comment every now and then in my book, meaning that I’m happy with the experiment.
Another page full of these numbers. Thursday, August 26th, with measuring the voltage gradient along the gel. Pages of this. [00:40:00] Friday, August 27th, a page which must have 100 numbers on it, measuring the voltages. Monday, August 30th, even more numbers. Let’s see, the one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15 — 17, 18, 19, 20, 21. Say, 20 rows, each with one, two, three, four, five — eight, nine, 10. About 20 rows with 20 entries every row, so that’s 400 numbers on this page, all written in by hand of what the voltage gradient was along there. Several pages of this sort, even there. Another massive one, September the 1st. I did even more measurements on it, because the time now is being measured [00:41:00] at one minute, three minutes, five minutes, seven minutes, 10 minutes, 15, 20, 25, 30, 40, 50, 60, 60 minutes in measuring the voltage gradient every minute or two in that. I must have been working like hell.
Still haven’t realized properly what is happening, until Tuesday, September 7th, when I begin to realize that it’s the voltage gradient is what’s causing the sharpness, not the voltage itself, so that these little measurements, these short distances, are really a measure of the derivative of voltage with respect to distance. You might say [00:42:00] DV by — or delta V by delta X, the voltage gradient. And the sigmoidal curve of down and then up when integrated gives a more or less Gaussian distribution, so that at the back of the gel, at the back of the albumin, the voltage gradient is lower. And at the front, the voltage gradient is high, but the voltage itself is going, following the protein, yeah. Words to that effect, I’m not sure it’s quite right, but that’s what I was thinking.[00:43:00] But I also was worried about the interaction of the protein, etc., with the copper electrode, and I have a comment, “Must try with an electronic volt meter to avoid polarization,” because my volt meter would cause some polarization. But I wasn’t that frightened of making measurements.
So in this book, the (inaudible) ends then to do some thinking about doing some experiments with heparin, though I don’t [00:44:00] see that I have any of that specifically. But use this technique for repeating heparin experiments with new heparin. So I must have used some heparin earlier that I didn’t notice. But the end is, that’s the last page in this notebook, which ends on September 10th — Friday, September 10th, 19-something. (laughter) No year on this. I remember that problem, actually, in trying to sort out these gels that I found at the notebook, nowhere had — had the year in it. What is it?
But it’s been entered afterwards in red crayon, is 1954 book three, that’s how we finished (inaudible).