I was exposed to Stein and Moore who were the reigning protein chemists in those days, and who had invented the amino acid analyzer, and who thought I was quite daft for trying to do something like antibodies. The issue is very simply put: Sanger and Tuppy had done the first brilliant complete analysis of what's called a protein... insulin, which had a molecular weight of 6000, that's 6000 times the molecular weight of hydro... the atomic weight of hydrogen. They, Stein and Moore, who, by the way, got the Nobel Prize at the same time I did in chemistry. Stein and Moore had been working on the amino acid sequence of ribonuclease, the very important enzyme, and I think the molecular weight was of the order of about 13,700. Here I was, going to do the sequence of a molecule that had a molecular weight of 150,000. If that didn't put me away in the loony bin, I don't know what. But I in fact stayed at it and I remember quite clearly experiencing what I'll call an epiphany, although that's too dramatic a word.
The... what intrigued me at that time, as I said, was physical chemistry, so I took my antibody fractions out of that Cohn fraction II, and I started treating them chemically with a variety of tricks. One of the tricks was to break the so-called disulphide bond, the sulphur... sulphur bond that occurred in certain proteins from an amino acid called cysteine, to make something called cystine. And I was very proud of myself because I made a rotating amperometric electrode that would measure how many of these bonds I broke. At the same time I was very intrigued – and here we go back to my fake, my disastrous welding machine, or brazing machine of my youth – I was very intrigued with the ultracentrifuge. In those days they had this giant instrument called the analytical ultracentrifuge where you could measure the so-called sedimentation constant, how fast the molecule fell in a field of 300,000 times gravity – a very, very high-spinning rotor which contained a little optical system that you could look in... invented by The Svedberg, a Nobel laureate in Sweden.
And so I was working at my amperometric electrode one day when I said, ‘You know, maybe I should block these sulphydryls that I make when I cleave these bonds, and stick it in the centrifuge.’ I had been of course looking at the native molecule in the centrifuge and that made a nice peak. When I did this it broke those bonds and put it in the machine. It didn't seem to move at all compared to the native molecule, so I rapidly made some mathematical calculations about maybe it unfolded and that increased the... the drag and the solute, and maybe that accounts for why it's so slow; it doesn't sediment. It turned out that nothing... nothing of that kind could explain the result. The only thing that could explain the result was that I had cleaved the molecule and that the molecule consisted of chains. Well, we'll come back to that, but the main thing was that... I can't quite call that an epiphany. I'll tell you about others that like are contrasted with, but gradually it became clear to me that what I had concluded was contrary to what was in the literature and that was of course extremely upsetting but it does teach you something about science and the way it proceeds – mainly, that there's a lot of stuff in the literature that doesn't quite gel even though it looks that way. So that was the beginning of my total absorption and of course there were several thoughts that were associated with this.
