How I became a physical biochemist – a Jack of all trades and master of none
This essay was written by John Eccleston and was first published in the 2009 Mill Hill Essays.
John Eccleston led a research group at NIMR from 1984 until 2008. After retirement he remained an active scientist until he was taken ill in June 2009. He passed away three months later. This essay is extracted from an article he was writing in collaboration with Professor H. Gutfreund FRS on ‘Changes in Laboratory Practice since 1945’. The article is remarkable but not unique in describing how it is possible to prepare for a career in science through hobbies, hard work, motivation and parental guidance by a route quite distinct from the conventional highflying university degree. But a second and equally special quality is evident in the last two paragraphs. Having achieved what must have seemed a dream goal of a doctoral studentship, he twice turned his back on the doctoral pathway he was on. Only at the third attempt did he find his Rosetta stone.
As I get older, I have started to look back at why I entered a career in science at the junction of biology, physics and chemistry. It was mainly due to serendipity but there were influencing factors. My interest in biology was aroused when my father introduced me to fish keeping; first goldfish and other coldwater fish, and then tropical fish. As well as the practical aspect of maintaining and breeding fish I developed an avid interest in their physiology, behaviour, classification and ecology. The culture and collection of ‘wild food’ led me to a wider interest in biology. One of my best presents was a toy microscope which revealed a new world to me. Other aspects of fish keeping involved physics (how a thermostat worked) and chemistry (concepts of pH and water hardness). The other hobby my father introduced me to was photography. Initially this just involved framing the photograph, pushing the button and then waiting for the photographs to come back from the local chemist shop. However, the acquisition of a contact printing set changed this. The set consisted of a cardboard frame, contact paper, developer, fixer and some sort of safelight. Watching photographs develop seemed like a miracle to me. Later I acquired more complex cameras and enlargers. Both fish keeping and photography played important parts later in my career although much of my photographic knowledge is now obsolete since the advent of digital technology.
My knowledge of physics was extended by trips to a nearby ‘war surplus’ shop where it was possible to buy small things such as switches, lights, buzzers, bells, rheostats and electric motors, which my father taught me to use in simple circuits. He also helped me to build a crystal set and one- and two-valve shortwave radios. Finally, my interest in chemistry came from a present of a chemistry set. I augmented this with chemicals purchased from the local chemist shop and from a laboratory supplier in central Birmingham. It was amazing, thinking of it now, what chemicals I could buy as a 12 -14 year old.
A prized possession was a ‘substitute’ Kipp’s apparatus, equipment designed in the mid-nineteenth century for gas generation. Although my version did not have the three glass spheres of the real thing, it worked on the same principle, that the increasing gas pressure of the product of the reaction caused the liquid reactant to be separated from the solid reaction, thereby controlling it. The reaction of iron sulphide with hydrochloric acid to produce the noxious evil-smelling gas hydrogen sulphide was a popular reaction, something that would now only be allowed in a laboratory fume hood. I also reacted zinc with hydrochloric acid to produce hydrogen and marvelled at the flame of the burning gas. However, it was a case of a little knowledge being a dangerous thing. The next morning I asked my father to light the flame again. However, air had obviously got into the apparatus and there was an almighty explosion. A hole in the plaster of the ceiling where the thistle funnel hit it remained for sometime afterwards as a reminder of my stupidity and of how lucky we had been.
It is probably best to skip over my school years since I only obtained passes at ‘A’ level chemistry and physics, surprisingly failing biology which was my favourite subject. I was therefore not able to take up a place on a Pharmacy degree course. This was most likely a good thing as I had spent four weeks working in the local branch of Boots and came away, probably wrongly, with the impression that the pharmacists just counted out pills, and the more senior ones became shop managers. I applied for a number of technician jobs in the Birmingham area. One was at Southalls, a manufacturer of women’s sanitary products. I think it involved sterilisation of products by radiation but I did not get the job. I also applied for a job at Davenports, a small local brewery. I was very interested in this, even though I don’t think that I had tasted beer at the time! I think that I would have got this job but I was told that, as well as the science, the person would be trained in all aspects of brewing and they didn’t think that my physique was up to rolling barrels of beer around! It is interesting to note however, that a lot of university biochemistry departments grew out of departments of brewing. Finally, in 1962, I applied for a technician position at the Midland Centre for Neurosurgery and Neurology, a small seventybed hospital in Smethwick. There were about fifteen applicants at the interview, for three technician posts in histology, biochemistry and EEG (electroencephalogram). Because of my interest in biology, I expressed interest in the histology position but I was offered, and accepted, the biochemistry position. I think that one of the reasons that I got the job was that the pathologist had a tropical fish tank in the department which looked rather putrid and contained a few sickly looking fish. One part of the interview revolved around whether I could get it back into pristine condition, how long it would take and how much would it cost! I spent the first few days at work doing this. Later on, I amazed the pathologist by curing one of the fish of a fungal infection!
I was now a biochemistry research technician working on a research project in the Pathology Department of Dr. A. Woolf. The aim of the project was to find the cause of muscular dystrophy, a wasting disease of the muscles. The main approach was to compare the intermediary metabolism of dystrophic muscle with normal muscle. I had to wait outside the operating theatre door to collect 100 mg samples of biopsied dystrophic muscle and samples of normal muscle taken during routine operations. These would be taken back to the lab on dry ice and incubated in a solution containing radioactive glucose in a Warburg apparatus. This equipment enabled carbon dioxide output to be monitored and to show that metabolism was occurring. The muscle strips were then extracted in acid, neutralised and the extract was analysed by two-dimensional descending paper chromatography. Chromatography at that time was a fairly novel but simple technique that provided a means of separating and analysing compounds that diffused at different rates on filter paper. We used a phenol solvent which left my fingers white (I don’t recall anyone in the lab wearing safety gloves). The chromatograph was then placed in contact with an X-ray film and left to expose for about three months before being taken to the X-ray Department for developing. The radioactivity shown on the film was traced onto the chromatograph and the spots cut out. I also ran a large number of chromatographs of standard compounds which were detected by spraying with one of a range of colour reagents. Ninhydrin was one reagent, which gave me my first of many experiences of purple fingers. About thirty of the radioactive metabolites were identified. The main conclusion of the work, published many years later, was that in dystrophic muscle there was more conversion to fructose than in normal muscle. Now we know this was an effect and not a cause of the disease. The radioactive compounds were from ‘The Amersham Group’- then a part of the government nuclear programme.
A second project, conducted almost entirely by myself, was to characterise and purify proteolytic enzymes from human muscle. These enzymes, which are themselves proteins, cause the breakdown of other proteins in a characteristic way. The rationale for this was that muscular dystrophy causes wasting of the muscle but the choice of topic must have had some connection with the fact that my supervisor’s doctoral thesis was on the proteolytic enzymes of a fungal pathogen. The first step was to go to the mortuary during a post mortem examination and wait while the pathologist provided a muscle sample. The sample was then transported back to our 5°C cold room, where it was worked up. This was my first experience of an environment where I would spend a lot of my later career. The muscle was homogenised in a blender and centrifuged in a refrigerated ultracentrifuge to separate the important liquid component from solid material such as cartilage. Proteolytic enzyme activity was determined by incubating equal amounts of the extract with casein, a protein found in milk, for a certain time then precipitating with acid and, after centrifuging, measuring optically the amount of certain amino acids released from the protein. This was done at about twelve different pH (i.e. acidity) values so that plots of activity against pH could be made. My memory is that there were three major peaks of activity.
I then attempted to purify these enzymes. As was the usual practice I constructed purification tables showing at each step total protein, enzyme activity, yield and purification factor. The ‘bible’ that I used at the time were the four volumes of Methods in Enzymology that had just been published. Never did I imagine that this series would over the years run into hundreds of volumes and that I would co-author two chapters in it. My memory is that in these purifications, the yield of protein fell so much at each step that I finished up with vanishingly small amounts with insufficient protein or activity to measure accurately measure. It was not clear to me how this approach could be used to study the enzymes from dystrophic muscle biopsies where only small amounts of muscle could be used as a starting point. I did however develop a colorimetric assay for an enzyme, amino tripeptidase, which I was encouraged to write up as sole author and it was published in the journal Biochemica Biophysica Acta.
Considering that it was all a ‘one man and his dog’ operation, the laboratory was extremely well equipped thanks to funding from the Muscular Dystrophy Association. It contained a scanning spectrophotometer, scintillation counter, preparative ultracentrifuge, fraction collector, an outside cold room and a Tiselius apparatus for the electrophoresis of proteins. The lab also had excellent glass-blowing facilities. Our department’s Consultant Biochemist had a project to measure cerebral blood flow, which involved determining the level of nitrous oxide (laughing gas) in blood. He had constructed a very complex glass apparatus for which he had done a great deal of the glass blowing himself and he taught me the skills. Thin layer chromatography was coming into use at this time and I constructed a ‘spreader’ to make my own plates on glass though it had limited success.
My photography skills also came in to use here. As it was a small hospital, there was no full-time photographer so the chief pathology technician was responsible for undertaking photography work. However, when he was on holiday, I deputised for him. Although it did involve some work with patients, it was mainly pathology specimens using a plate camera. On one occasion the chief neurosurgeon, who ran the hospital like his own personal fiefdom, came storming into the lab asking for someone to photograph the filthy ceiling outside of the theatre to assist his complaint to the authorities. Someone had started cleaning it but by printing the photograph on hard paper, I was able to make the dirt appear worse than it actually was, so that the surgeon was very pleased with it. It didn’t need the advent of digital photography to ‘enhance’ photographs.
During my four years at the hospital, I attended college for a whole day and two evenings each week. On the advice of our Consultant Biochemist, I chose the route to qualify with the Royal Institute of Chemistry instead of studying for the Institute of Medical Laboratory Technology qualification like all the other technicians there. First I did the Ordinary National Certificate in chemistry at the Matthew Boulton College in Birmingham. This was in an old building in its final year of use and the facilities in both the lecture rooms and laboratories were poor. It was a very cold winter and the wash basins in the toilets froze over. When the thaw came there were buckets down the corridors to collect the dripping water! I then moved to the Chance Technical College in Smethwick which was housed in a modern building, and where the lecturers were all enthusiastic teachers. After two years there I gained a Higher National Certificate in chemistry. After a further year I passed the Part 1 Graduate Membership of the Royal Institute of Chemistry (Grad.R.I.C.) which was accepted as the equivalent of a pass degree.
I could have spent another two years at the college to obtain Part 2, the equivalent of an honours degree, but decided I had had enough of day release and chose to do a full time year at the Liverpool College of Advanced Technology which had a high reputation at the time. It later merged with several other colleges to become a Polytechnic which then became the Liverpool John Moores University. In 1966 it was a modern building, again with enthusiastic lecturers. As well as a heavy lecture load, the course was very much based on practical skills. There was a half day of practical physical chemistry and a whole day alternating between inorganic and organic chemistry. The latter took place in laboratories housed in an old warehouse in the docks area.
Inorganic practicals involved quantitative and qualitative chemistry, both based on classic books by A.I. Vogel. We soon discovered that the ‘correct’ answer to an analysis problem was the average of everyone’s results and after a few weeks of wildly varying results the spread became much narrower. Qualitative analysis involved first doing a Lassaigne fusion test to determine the elements present in the unknown sample. The sample was heated with sodium metal in a test tube until it was molten and the tube was then plunged into water in an evaporating basin and quickly(!) covered with a piece of gauze. Although we wore lab coats at this time, I remember having carried out the same procedure at school wearing blazers. After filtration, the filtrate was subjected to ‘group analysis’. An early stage of this involved hydrogen sulphide from a Kipp’s apparatus. Although this was in a fume hood, fifty students using it resulted in the whole lab reeking of hydrogen sulphide.
For organic practicals we were required to synthesise substances from simpler starting chemicals, or to identify the components of a mixture of two compounds. To identify components we first separated them by fractional distillation, crystallisation or solvent extraction. Functional tests were done on both compounds and finally, two derivatives were made of both, recrystallised and melting points determined. Consulting lists of melting points of the different derivatives allowed the identification of the original mixture. All of the organic practicals involved fifty students refluxing or distilling organic solvents in an open lab. Even though I wore a lab coat, the landlady at my digs could tell whether I had been doing organic or inorganic practical from my smell! The apparatus we used was basic, but the practicals were very instructive. Although we were working within guidelines we had to think what to do next in any practical rather than just following a recipe, which was later my impression of university biochemistry practicals.
At the end of the year, following the theory exams, there were two seven hour practical exams in both inorganic and organic chemistry. Apart from the exhaustion, I only have two memories of these. One was as I had started refluxing a solution of ferrocene, I remembered to put in a few pieces of porous pot but I was too late and a lot of the solution shot out of the apparatus over the bench and me, considerably reducing my yield. The other was an unknown, black, crystalline inorganic compound which afterwards discussion between the students gave a wide variety of positive tests for the elements but it turned out to be sugar charcoal which should have given no positive results. The only person to get it right was a student who had previously worked at Tate and Lyle!
Grad.R.I.C. results were not classified but I must have done reasonably well because I was offered a Ph.D. studentship by the head of department in organic chemistry. I decided instead to return to the Midland Centre for Neurosurgery and Neurology. I was now a graduate biochemist and as such was allowed to call senior members by their first names, and to have coffee and lunch in the doctor’s dining room instead of the technicians’ room! I started work on a project to measure intermediary metabolites of muscle by enzymatic analysis based on the recently published book Methods of Enzymatic Analysis by Bergmeyer. I registered as a Ph.D. student in the Biochemistry Department at Birmingham University. However, things did not turn out as I expected and in 1968 I moved to the Birmingham Children’s Hospital, transferring my Ph.D. registration to the University Chemistry Department. I was employed now as a Clinical Biochemist, paid for by the NHS. I was therefore required to learn methods of clinical biochemistry and to be regularly ‘on call’ at nights and weekends. However, my impression was that my main work was to do clinical research.
The object of my project was to study a group of inherited diseases then called gargoylism which involves deficiencies in lysosomal enzymes. This involved analysing samples of urine of normal and affected patients. After only a couple of months, because of personnel problems, I was put in charge of the ‘routine’ lab for 50% of the time, alternating with a senior technician. Capillary blood samples were taken from children on the wards by the technicians and analysed by various methods. Urea, bilirubin, glucose, protein and various enzymes were analysed by colorimetic methods, sodium and potassium by flame photometry and chloride by an electrometric method. The most technically difficult method was that called an ‘Astrup’. The pH of capillary blood was measured. The blood was also equilibrated with 4% and 8% carbon dioxide and again the pH measured. The levels of carbon dioxide and bicarbonate in the blood could then be calculated. All transfers of the clinical samples were done by mouth pipetting, this being before the emergence of HIV. Automated methods were beginning to be used at the time in many clinical biochemistry labs. Samples were automatically sucked up into a flow system separated by bubbles, mixed with the appropriate reagent and passed through a flow system at the appropriate temperature and time before passing through a colorimeter. However, since it was a children’s hospital, mainly only capillary blood was collected, too small for such automated methods, and so nearly all analyses were by manual methods. Although I was doing routine work for nominally only 50% of the time, because of holidays, sick leave and the usual emergencies and crises it was in fact much more than that, leaving little time for my research project. I therefore applied for, and gained, the position of Research Assistant in the Biochemistry Department at Bristol University, and my career as a full time physical biochemist began.
Within a year John had registered at Bristol University for a Ph.D. degree. In his thesis, again in the area of muscle, he laid some of the foundations of the chemistry, kinetics and energetics of muscular contraction. After five years he went to the University of Pennsylvania where his interest turned to understanding the mechanisms that regulate biological processes, and how energy is used to effect these mechanisms. In 1984 he joined the MRC National Institute for Medical Research as a Research Group Leader. He published many papers including in 1997 three papers in the journal Nature – one of the highest rated science journals worldwide, and one in which most scientists would be pleased to publish once every five years. In his later years he made important contributions to multidisciplinary studies bringing his biophysics and chemical expertise to bacteriology, virology and malaria.