I`d love to hear about the early days, the beginning, when you began protein sequencing and how did you come to feel passionately that you wanted the sequence proteins? Tell me, why did you come to feel passionate about sequencing proteins in the first place?

Well passionate`s a bit of a big word I think, I don`t think it was really like that at all. I was getting a job really. This is about 60 years ago you see. I got a job with Professor Chipnall at that time, after I`d finished my PhD. He was a protein chemist and he was essentially involved in amino acid analysis, just finding out the number, the amino acids which were present in any protein, and he happened to be working on insulin at the time. He was interested in the pre-amino groups of insulin and he really gave me the problem.

I think when you start out research you don`t really have great passions. I mean I wanted to do research and I was interested in biochemistry, and I think I would have settled for a job really. So I don`t think there was a great passion, but looking back, it seemed the sensible to do actually. At that time proteins had been discovered, but there was very little known about their exact chemistry. They knew the amino acid composition generally and that was about all. So I joined this group and it happened that insulin was one of the few proteins that you could get in a pure form in those days. It was something of course that was of biological interest and medical interest.

So I joined this group working on insulin with Tidnall and his colleagues, and noticed that there were a very high proportion of free amino groups in insulin. Those are the groups at the ends of the chain. And of course there are other amino groups which are lycene residues. Lycene you know is an amino acid with 2 amino groups and just one carboxyl group. The only amino acid that has an amino group on the side chain. So he suggested that I work on it. I looked at various methods which had been used for this type of thing but the substance which I finally came up with was this DNP compound, di-nitrofluorobenzine. That reacted with amino groups and you could then treat the protein, treat the insulin with GNFB… FDNB actually it was, and that would couple onto the free amino groups of the protein. I think the important thing about this compound was that it was coloured so you could see, and of course that was very important in those days, you didn`t have fancy methods for detecting other types of compounds. So what I did essentially was to treat the insulin with the DNFB and then hydrolyse with acid, and this shows what happens, here you have the end of the chain, that`s an amino acid chain. I think you know what a protein chain looks like.

We`ll come back to that because it`s important but the DNP sits on the end of the chain here?

The DMP sits on the end of the chain and of course it`s on the lysine residues too, if there was a lysine there that would be a DNP there. So then you hydrolyse with acid and you split all the peptide bonds and you get a lot of free amino acids and you get some coloured substances.

And according to exactly where the chain splits they come in different sizes? Is that right?

We haven`t got to that … First of all you hydrolyse with strong acid - you just get the DNP amino acids and then of course the main problem at that time was how do you separate them from one another? You see chemistry, at that time was usually done by re-crystallisation and distillation and very crude methods compared with what we can do now. But it just happened that at that time a very rapid and useful technique had been developed by Martin and Singh, the method of partition chromatography.

You were saying that the Martin and Singh partition chromatography was critical at this stage?

Yes, well this was a new method. That was invented essentially by Martin, who is a very bright person, he was a sort of physical chemist and rather eccentric person. He invented this method which you could use it first of all for analysing proteins, making the acetyl derivatives and then you could put them on a column and essentially separate them out, one from another and they come out of the bottom and you collect it, the first sort of chromatography. Chromatography at that time I think was largely absorption chromatography with coloured dyes but this method was applicable to almost any substances and it was particularly good for the acetyl amino acids and it happened that in the lab Tristram, one of Tim`s students, was involved in using this acetyl method and separating amino acids because you could analyse for some of the amino acids which were very difficult to analyse. So he was using it for analysis on the acetyl and of course it was very good in fact for these DNP derivatives. They behaved a bit differently so you had to find a different solvent to use with them.

Because this is a very sort of oily sort of group, is that right? You need special things to…

Yeah that`s right, it`s more a sort of soluble in organic … And of course it was covered so you could see these lovely yellow bands coming down the column and collect them. So that was a big thing really in all this work and it made it possible to identify the amino acids which were present. We found 2 DNP derivatives, DNP glicine and DNP phenalalanyne…

And those were the ones that were at the ends of the chain?

Those were the ones at the end and we got some epsilon DNP lysine, where the lysines had been but that was quite different because they`d had an amino group and a carboxyl group and it was water soluble, whereas these were …