The 1st Floor, Department of Biochemistry,
University of Cambridge (1952-58)
by Brian S. Hartley
I joined the Department in 1952 by happenstance, in what I believe were its halcyon years. I was awarded an ICI Fellowship to work with Keilin in the Molteno Institute, so had mugged up his seminal papers on cytochromes. At our first interview I therefore gave him a spiel on a project to chemically modify cytochrome c so as to find out which groups interact with cytochrome b. Keilin, a small man with a pronounced German accent, smiled kindly on me and asked “That’s not you really want to do is it Hartley?” I confessed that I really wanted to work on the mechanism of action of chymotrypsin which had been my PhD topic with Bernard Kilby in Leeds. He said “I thought so. This is not the best place to do that, so I have spoken to Malcolm Dixon and he has agreed to give you space in his Enzyme Unit on the First Floor of the Biochemistry Department”. Such generosity in giving away a prized Fellowship which has been awarded to his Department was typical of the great Cambridge scientists that I met subsequently.
I discovered that the First Floor was crammed with these, Malcolm Dixon showed me to a bench in the North lab. where his students and postdocs worked. Central pride of place was given to a large refrigerated open tank of ethanol in which his student Vince Massey was purifying flavoproteins by techniques developed by Bob Morton. Malcolm, a large shy man, hesitantly asked me to avoid knocking out my pipe against the walls of this tank, but noting that Vince was smoking a pipe at the adjacent bench I felt free to indulge this bad habit at my own bench. Vince continued his research on flavoproteins throughout his subsequent career in Sheffield and at Ann Arbor, Michigan, and so became a world expert in the field and Fellow of the Royal Society.
Malcolm was interested in the kinetics of the ‘nitrophenol burst’ that I had discovered in my PhD work when reacting chymotrypsin with nitrophenyl acetate. This implied that the active site serine was actually acetylated by the substrate during the catalytic hydrolysis (though my first ever paper was rejected by the Biochemical Journal for suggesting this heretical concept). However I was not longer in love with enzyme kinetics, so Freddy Gutfreund agreed to pursue that problem in the Department of Colloid Science in Free School Lane. He too eventually became an FRS for his development of rapid enzyme kinetics techniques.
I had come to believe that only knowledge of enzyme structure could explain enzyme mechanism, although there was no obvious way to acquire this. However next to Edwin Webb’s little office from which he seldom emerged from drafting his seminal book with Malcolm Dixon on enzyme kinetics, was a little darkroom. In this an amiable Argentinean, Gregorio Weber, was inventing and constructing novel machines to use fluorescence to study protein conformation. Using modifications that he had made to Einstein’s equations of Brownian motion he was able to deduce protein size and shape and changes therein, using a range of fluorescent dye labels for this purpose.
Gregorio became my hero because his ideas were always entirely original and way ahead of the field. I was therefore shocked to read an exact copy of his results of serum albumin structure in Federation Proceeding following a recent visit by an American scientist, who shall be nameless. I rushed to show this to Gregorio expecting a burst of fury, but instead he smiled to me and said, “Relax Brian. It doesn’t matter. This man, he is a little man. For me, (tapping his forehead), there are more ideas where these came from”. And so it proved.
I was amazed that Frank Young couldn’t find a post in the Department for this near genius, so Gregorio went to the old cinema in Sheffield where Quentin Gibson had succeeded Hans Krebs, and Vince Massey soon followed him. But once again the University let Gregorio down by refusing to give him a personal Chair. ”No one can be any good if he wants to stay in Sheffield”, the Vice Chancellor said. Taking him at his word the whole Department therefore resigned and took up posts in the USA.
Vince and I used one of Gregorio’s fluorescent dye labels, “Dansyl-chloride”, on chymotrypsin and chymotrypsinogen to see if there was a difference in conformation. Not entirely to my surprise it reacted rapidly with the active centre of chymotrypsin to give a green fluorescence whereas it reacted slowly with chymotrypsinogen to give a yellow fluorescence typical of that given by labelled amino groups. (With Bill Gray I subsequently developed a protein sequencing strategy using this reagent). We were also excited by the observation that the fluorescence of Dansyl-chymotrypsin gradually changed from red to green implying migration from an adjacent histidine to the active site serine, would explain the pH dependence of the catalytic activity. “Red to green means histidine” I used to chant, and I got the urge to become a protein chemist to discover which histidine was involved.
I plucked up courage to ask Fred Sanger ("three labs down the corridor” to teach me his technique of end-group labelling with fluorodinitrobenzene (FDNB). Vince and I found that it labelled the N-terminal Ala- and Thr- of native α-chymotrypsin but not the Ile- of the B-chain. This implied a change in conformation after activation of chymotrypsinogen causing the latter to become buried (as proved to be the case when the structure was determined by David Blow’s group almost 20 years later).
I fell in love with protein chemistry after this first brush, and conceived the absurd ambition of determining the complete amino acid sequence of chymotrypsin as a first step to elucidating its structure. Edwin Webb, Malcolm and Gregorio all warned me that this was impossible for one person since large groups in Seattle and Prague were already working on the problem. However Gregorio finally looked me in the eye and asked “Do you really want to know the answer?” I had to ponder for a minute; then said “Yes”. “Then do it.” He said. So I did – though it took another 15 years.
The secret of Fred’s protein chemistry techniques lay in a small room in the basement in which one suspended sheets of wet paper in tanks of petrol containing pyridine and passed a 3000 v. current through these. After drying the paper, one then suspended it in a trough of phenol in aluminium tanks. I was not concerned about the health hazards because in Leeds I had synthesised enough nerve gas to kill the whole population of the city by bubbling phosgene through phosphorus pentachloride (both deadly chemicals in themselves). However it is interesting to muse that Fred Sanger would never have been allowed to earn his first Nobel Prize in any laboratory today!
At that time Fred was well on his way to completing the complete sequence of pig insulin and others in his group were working on insulins from other species. I was thrilled to learn that pigs were more closely related to cows than to sheep so that amino sequences could reveal evolutionary history, and I held this enthusiasm for the rest of my career.
But Fred himself went off on another tack, trying to develop methods to determine partial amino acid sequences in phosphoproteins using radioactive isotopes. Ovalbumin was his chosen model, so he designed an experiment to label it in vivo by feeding a chicken with almost a Curie of 32P-phosphate. Such a quantity of radioactivity had never been seen in the Department before so the diligent Radioactivity Safety Officer, Geoff Brown, rigged up an impressive wall of lead bricks in a little room under the stairs in the Basement. The chicken was perched high up and totally shielded by these, but a spiral ramp below the nest allowed the egg to roll down into a lead-shielded flask below. We were all told to contact Fred and Geoff if the chicken started to cackle, so I raised the alarm when I heard that noise from the nearby high voltage electrophoresis room. Fred had calculated that the radioactive content of the egg would be well within the safety limit, so equipped with monitors we watched as the egg eventually emerged and rolled down the rails. But the monitors screamed and Geoff Brown nearly fainted. “Damn!” said Fred, “I forgot the shell” (which is about 25% w/w phosphorus).
Fred was therefore amenable to a much safer experiment in which his student Mike Naughton and I labelled the active sits of trypsin, chymotrypsin, elastase and subtilisin with 32P-DFP and then did a partial acid hydrolysis of each. The radioactive patterns after electrophoresis showed that the active sites of the former were identical whereas subtilisin was quite different; proving that Fred’s concept of such techniques was both valid and useful. Later, however, Mike’s laboratory in Canberra unfortunately burned down due to a fire in the high-voltage electrophoresis laboratory, so these techniques became less popular.
By this time César Milstein had joined Fred’s group to study the active site of phophoglucomutase by such techniques, and we were all contemplating an alliance with Max Perutz’s Cavendish Laboratory in a new Laboratory of Molecular Biology. There was a push as well as pull for such a move because it was clear that Fred’s group could not expand much further in the limited space of the Biochemistry Department. Indeed Frank Young told me so face-to-face when I applied for a vacant Demonstrator post in the Department. “I should tell you frankly, Hartley, that there is no future for you in Cambridge.” He said. But Fred had other ideas and offered me a post in his LMB group after my return in 1960 from a two year Whitney Fellowship in Seattle working with Hans Neurath.
I had already established contact with the Cavendish Group after I discovered enormous crystals when doing ammonium sulphate precipitations of DIP-chymotrypsin. These were quite different in shape from the normal rhomboids so I took them to Max who passed me on to his student David Blow. David showed real interest and began his long grind to determine the structure of chymotrypsin, which was the beginning of our lifelong collaborations in the MRC Laboratory and later at Imperial College on tRNA synthesises and glucose isomerise.
There were lots of current or future FRS’s and Nobel Prize winners on the First Floor at that time. Sam Perry was studying actomyosin in the lab opposite Malcolm Dixon, and Joe Needham, the “Reader in Oriental Biochemistry” and his wife Doffy Needham had an office at the corner of the stairs. Next came Robin Hill in the lab opposite to Fred Sanger. I had already heard about Robin’s capacity to pull legs when he approached me as “an expert on redox dyes” which I was using for a Practical class experiment, so I was able to reply, “No, Dr. Hill. You are the expert.” Robin became my neighbour when we moved to Barton because he had a small farm there which he used for growing nettles. “The locals think I’m crazy”. He said “but I get paid a fortune for them by the ARC for making chlorophyll.”
I saw Robin in top eccentric action when he was asked to follow Calvin’s epic presentation of the dark reaction in photosynthesis at the 1953 Brussels International Congress of Biochemistry. This was in a huge tall steep Anatomy Theatre, and Calvin showed a series of brilliant colour slides with exquisite timing. However, when the Chairman called on Robin to comment on the Light Reaction, he came slowly down the steps from the very back of the theatre, turned nervously to face the huge audience and slid from side to side with his back to the semicircular Lecturers bench. He then walked over to the Chairman in the front row and asked audibly, “How do I get in?” The Chairman lifted a flap and Robin then started to take a tin box from his pocket, lifted a layer of cotton wool and then removed his watch which he laid on the bench. “I sometimes think that the chloroplast is like a photoelectric cell” he began, walked over to the blackboard and drew two vertical parallel lines in chalk. He then turned to the audience and asked loudly, “Can you resolve these lines at the back?” After murmurs of assent he drew an arrow and said, “This is the light” ….pause…..”I forget which is positive and which is negative?” Then he leaned over to Calvin who was mopping his brow in the front row after his efforts, and asked, “Do you know, Calvin?” Calvin shook his head. “I think this is positive.” Robin continued happily.
But my best Robin Hill story is second-hand, told to me by Major Letham who had recently arrived as Departmental Superintendent. As he entered the Downing Site from Downing St. he encountered Robin dawdling under the arch who said to him, “I’m Robin Hill. You’re Leytham aren’t you? May I walk with you?” “With pleasure.” Leytham replied and they walked side by side to the foot of the Botany stairs. “After you, Letham”, said Robin. Somewhat nonplussed about where he was going, Letham said, “No, after you Dr. Hill”. They climbed the stairs and chatted while walking along the main corridor turned left and emerged through a back door opposite an entrance into Agriculture. Once again Robin performed the same routine but this time took Letham downstairs through the basement full of pig pens and up some iron steps to emerge by the Biochemistry back door. Now knowing where he was, Letham agreed to enter first, but when they were both inside Robin asked, “Do you always come this way, Leytham?” “No, I’ve never been this way before.” He answered truthfully. “Neither have I!” said Robin.
The large South Laboratory housed Don Northcote, who appeared perfectly normal though hew worked on plants about which I knew absolutely nothing, but also another obvious eccentric with long hair and sandals which were very Bohemian at that time. Despite his appearance, Peter Mitchell turned out to be a decent chap with a fondness for draught Guinness which we shared regularly in a small bar by the side of the Guildhall, decorated like a Methodist chapel.
We had long arguments about how molecules get across membranes. Peter had crazy ideas about static electricity, thermodynamics and holes in membranes that he called ‘porters’ (a tribute perhaps to the black brew before us). I tried to persuade him that this could in no way explain the specificity of membrane transport, which must involve a ‘swing door mechanism’ in which ATP was used to rotate binding sites across the lipid bilayer.
To prove me wrong he thereafter regularly sent me copies of his publications and I became sort of neutral referee in his battles with the orthodox Lehninger/Slater school of oxidative phsphorylation. He left to set up his own private laboratory in Bodmin (being an heir to a great fortune) and stole Malcolm Dixon’s right hand assistant, Jennifer Moyle, to help him. I met him thereafter by chance in a compartment in a train to Exeter where I was to conduct a PhD oral and asked him how we could possibly afford to pay assistants out of his own pocket. He explained that he paid them very low wages. “I give them medals instead,” he said, reaching down and opening a heavy briefcase. Inside were dozens of huge solid gold coins embossed with Peter’s face. “I make these myself,” he explained. “They can nearly double their salaries with them but they pay no tax.” Soon after I was proud to receive a signed copy of his privately printed little grey book on the chemiosmotic hypothesis – a tribute I believe to the ‘porters’ that we had shared in the past.
I too left Cambridge in 1958 to take spend 2 years with Hans Neurath in Seattle to exploit ion exchange methods for peptide and protein purification developed by my friend Herb Sober. I returned briefly to the Department in 1960 to a bench in the attic where my first PhD student, Bill Gray and I developed the Dansyl-Edman technique for peptide sequencing. However, I had relatively little contact within the rest of the Department at that time because all our efforts were in liaising with the Cavendish group and in designing the new Laboratory of Molecular Biology which was then under construction. The breakthroughs in enzyme and protein structure and in DNA and gene expression that followed have become legendary. However, I hope that the above comments show that no small part of these successes had their origins in the First Floor of the Biochemistry Department.