Vallee

The arrival of the new millennium was the incentive to summarize the events which led to the establishment of the Biophysics Research Laboratory (BRL) and its successor, the Center for Biochemical and Biophysical Sciences and Medicine (CBBSM) at the Harvard Medical School.

This very personal account makes no claim of being either complete or comprehensive. It is a memoir and not a history of either the science or views of its time. However, it has given me pleasure to recall people and events which were instrumental in shaping my professional and personal life. Most importantly to me, perhaps they recalled the events and individuals who taught and guided me and ultimately led me to focus on metals in biology as my life's work.

My academic career began at a time when it was difficult and expensive to obtain a concurrent education in both science and medicine, and the number of individuals who did was relatively small. There was little, if any, effort on the part of American universities to facilitate the process and/or avoid repetition and duplication. The undertaking was unusual and expensive both in terms of time and effort spent. Few could afford it, and I was one of them. Certainly, the rewards for trying were not compelling then. Yet, there also were indications that the expansion of the scopes of physics, chemistry and biology would be rapid and that interdisciplinary mergers would be in the cards. Once this would occur, it would call for backgrounds that would encompass multiple disciplines. Suitably educated individuals could then become champions of borderland areas of the emerging fields yet to be defined and named.

It was my good fortune that the government assigned me to a wartime project at one of the most active and vital scientific interfaces among these disciplines during World War II. The National Research Council sparked a number of inter-institutional arrangements among Harvard Medical School, especially its Department of Physical Chemistry, the then Peter Bent Brigham (now Brigham and Women’s Hospital), and the Physics and Biology Departments of MIT. Inter-institutional arrangements were then most unusual, and those that were made were the consequence of the World War II effort. These collaborations were intended to advance both basic and applied sciences in the solution of myriad wartime questions and problems, which the government deemed to be in most urgent need of solution. Among these were e.g., the storage and preservation of blood for transfusions; the procurement of blood plasma to be made available in quantity for the treatment of surgical shock; the isolation, characterization and synthesis of antibiotics such as penicillin and sulfonamides, to name but some of the prominent issues and questions that university laboratories all over the country were addressing encouraged, solicited and financed by the government.

I was assigned to the HMS/MIT team which collaborated with the HMS Department of Physical Chemistry under the direction of Drs. Edwin Cohn and John Edsall who pioneered in the isolation and characterization of blood proteins. They used the isotopes then produced by the MIT cyclotron (prior to the advent of nuclear reactors) for collaborative biochemical experiments performed at HMS. Physical measurements of isotopes were performed in the Physics Department at MIT, and I was made a go-between it and HMS.

The accomplishment of the wartime project on which I served is a matter of record which I shall not address here. It was a remarkably successful undertaking that accomplished its objective and led to the prolonged storage of blood which is now commonplace. My role in this was minimal, but it served me as a most remarkable apprenticeship for my ultimate career; I remain grateful to all those many that taught me.

It was also a most rewarding intellectual assignment which exposed me to a broad range of physical and biological sciences and departments which were undergoing major changes and expansions under leaders who were generous with their time. I was encouraged to use mine expeditiously.

Over the years I carried a full load of relevant MIT courses in nuclear physics, optics and spectroscopy, biophysics, organic, inorganic and physical chemistry as well as instrumental analysis. In addition, there were specialized courses of the MIT Biology Department in physical biochemistry, electron microscopy, x-ray diffraction and newly emerging optical techniques such as optical rotatory dispersion and circular dichroism which had moved to the then forefront of biology.

Importantly, from my point of view, there were no charges for the education. I was allowed to audit any and all courses that my time and endurance permitted, providing what was for me the most critical and major benefit, a real windfall! Over and beyond my work on blood fractions which involved hemoglobin, I became very interested in the metabolism of iron and that of other metals such as zinc and copper, about the occurrence, localization and function of which little or nothing was known at that time. Spectrochemistry was developing rapidly, and I became aware that physicists, however, were moving to nuclear physics leaving spectroscopy to individuals who were largely trained in physical, organic or inorganic chemistry initially, but biologists were not among them. Certainly, physicians were conspicuously absent from that group.

With the end of World War II in sight, it became necessary for me to make concrete plans for my future.

I had become very intrigued with the intellectual and research potentials of spectroscopy in general and emission spectroscopy in particular, stimulated to no small extent by the pervasive role of iron in hemoglobin structure and function. Days in the library soon confirmed that knowledge of inorganic biochemistry and physiology was most prominent by its absence and with seemingly limitless opportunities for exploration of their biological role(s). Atomic spectroscopy seemed like an almost ideal portal of entry for the quantitative study of metals and their metabolism, and MIT looked like an ideal place to do it. This was an exciting discovery all around, and it did not take me long to come up with a plan. I decided to make the detection, structure and function of metal containing biological systems (proteins in general and enzymes in particular) the pivot of an extended research plan. Clearly, emission spectroscopy would play a central part in it.

I had become aware that the Committee on Growth of the National Research Council had just then established fellowships to support two incumbents selected from the country at large for three years each to undertake basic research in biology in fields of the applicant’s choice and in an institution willing to house him. This then unique fellowship may well have been the result of the wartime experiences just recalled in my above remarks. I was lucky to be selected for one of them to be spent in the physics and biology departments at MIT under the direction of Professor John R. Loofbourow, professor of biophysics, and Professor George R. Harrison, professor of physics, head of the MIT spectroscopy laboratory as well as dean of science. My application essentially called for continuation and extension of the wartime insights in the direction implied above.

I was awarded this three-year fellowship in 1951, but it soon turned into six years which I spent at both MIT and HMS (PBBH) in a joint appointment. For the second half I was ultimately supported by the Howard Hughes Medical Institute which was founded just then. The two successive fellowships thus coincided with the establishment of the Biophysics Research Laboratory (BRL) at HMS where the work that I initiated at MIT continued, now emphasizing the design, construction and utilization of new spectrochemical facilities and their impact on biology and medicine as well as other disciplines.

The famed spectroscopy lab of MIT had served the Comptons and George Harrison well and gave me space and facilities to develop emission spectroscopy using the expertise and facilities of the Biology and Physics shops and having the advice of John R. Loofbourow and James Archer, then a young professor of physics at MIT. These relationships continued for many years and beyond the early times which I described. In fact, with the expert help provided by the shops I built a flame spectrometer by conversion of a one meter Wadsworth grating spectrograph into a flame photometer designed to detect and quantify Na, K, Mg and Ca in biological matter. At that time these elements could not be measured accurately when present in physiological concentrations. This new instrument would be prototypical of others ultimately capable of monitoring these elements routinely for clinical purposes. These elements were soon discovered to be essential for the diagnosis and treatment of seemingly infinite numbers of clinical problems in biochemical metabolism. In fact, these elements and the instruments monitoring them have become indispensable in the management of a multitude of diseases and are now employed routinely. The Jarrell-Ash Company of Newton ultimately also manufactured such an instrument.

Simultaneously, I continued efforts to define, develop and evaluate new spectroscopic flame sources for the excitation and spectral emission of atoms, e.g., the cyanogen/oxygen fuelled flames. The results of that work had broad implications for many other scientific disciplines as well as geology. I became conversant with the then current literature on flames and their characteristics which ultimately became crucial for the study and characterization of rockets.

At this point it seems best to include the reference to a reprint of a manuscript that I was asked to write for the Scientific Monthly, a journal that has long since merged with Science and, hence, does not exist anymore. My application for the NRC fellowship was based on the material presented there and describes the then state-of-the-art and knowledge in the field as well as my plans for future work. (Bert L. Vallee, The Function of Trace Elements in Biology, Scientific Monthly, LXII, 368, 1951)

My design of the Biophysics Laboratory to be established at HMS and its ultimate objectives were based on the utilization of spectroscopy in biology as the dominant contribution of the then physics to the enterprise.

This synopsis of my postgraduate education has said very little so far about the potentials of emission spectroscopy that I recognized at MIT. Some knowledge of the prior developments in that field is useful to appreciate the implications of that orientation in the development of the BRL.

The establishment of this laboratory and definition of its objectives was a major, policy initiative on the part of George P. Berry then the Dean of Harvard Medical School. He successfully solicited the Rockefeller Foundation, then under the direction of Dr. Warren Weaver, to found and support a laboratory to investigate the potential of spectroscopy in the exploration of metals and their function in biology and medicine. Dean Berry decided that the laboratory should be housed in the Hospital to emphasize the recognition on his part and that of the Rockefeller Foundation of the urgent need to merge the mushrooming knowledge in the basic sciences with the never ending demands for its speedy application to the care of the sick. Medical biology, biophysics, molecular biology and seemingly endless numbers of other designations tried to capture and identify the spreading extension of efforts to extend the scope of biological research. Ultimately, whatever its content and operations, molecular biology became a designation that was favored by many and accepted almost universally as descriptive of the new look and direction. Information transfer and removal of undesirable intellectual barriers was and has remained the intent of the undertaking of the BRL and CBBSM over the entire period of their existence.

In effect, all of the laboratory’s undertakings were based on the developments in science pertinent to biology and medicine during the last century. One will better appreciate the significance and far reaching importance of the judgment Dean Berry made with the help of a synopsis of some cardinal baselines pertinent to this initiative.

Like many other important developments in physical science these, too, can be traced to Newton who recognized and deduced the existence and importance of the electromagnetic spectrum given a minimum of data. The array of wavelengths of photons absorbed or emitted by atoms or molecules characterizes each of these. The wavelengths may vary from trillionths of a millimeter to thousands of kilometers; jointly they constitute the electromagnetic spectrum. The wavelengths of atoms and molecules in fact are their “fingerprints” and identify the source of radiation which is either emitted (emission spectroscopy) or absorbed (absorption spectroscopy), the former characteristic of atoms, the latter of molecules by and large. For the present purposes the discussion is limited to that part of the spectrum that can be segregated from the balance by prisms or optical gratings. The observed spectra are continuous unless the radiation is made to travel through a very narrow slit to yield a monochromatic image in the form of a line. In 1817 Fraunhofer observed such lines in the light of the sun which since then have born his name. Forty-two years later Kirchhoff and Bunsen showed that a spectroscope will identify radiation of different wavelengths and establish its origin.

Historically, spectrochemical analysis has been based on either emission of radiation by atoms when excited either by sparks, DC arcs or flames. In contrast, absorption of radiation is observed by molecules in solution, at or near room temperature. Thus emission and absorption spectral analysis were operationally distinct and separate disciplines until 1955 when it was recognized that in flames atoms can both emit and absorb radiation. The process of atomic absorption, like that of emission, can be adapted to spectrochemical analysis. A century had elapsed between the time that Kirchhoff and Bunsen recognized atomic flame emission, and the insight was gained that Fraunhofer lines are proof of atomic absorption.

This insight became the basis for an extraordinarily sensitive analytical method that combines the potentials for successful applications to biology. Once familiar with this new awareness, we were able to move experimentally with great speed in the course of my fellowships.

At the time that I began my fellowship work at MIT, spectrochemical analysis had not advanced a great deal; to employ current day language, its practice was hampered by relatively unsophisticated instrumentation which was “not user friendly”. To make the point tellingly, there was no equipment manufactured specifically to serve biological or medical markets which had not been developed and recognized as yet. Hence, if one urgently needed an instrument, one had to design and build it.

Therefore, it was necessary to become intimately familiar with the principles which underlie the characteristics and choices of spectroscopic devices and their components 1) the sources of excitation of atoms or molecules, 2) the radiation dispersing elements such as prisms or gratings, and 3) the radiation receivers. From the beginning I gave close and detailed attention to the study of all three in order to become sufficiently familiar with them to make informed choices for my major aim: the determination of the metal content of biological matter; its quantitative analysis and functional biochemical and physiological potential. In point of fact, I spent all of my spare time and effort at MIT on these and made substantial progress in choosing, aligning and composing state-of-the-art equipment. Ultimately this resulted in novel combinations of sources of excitation, gratings and receivers which proved successful for the accomplishment of my objectives.

In the course of time I became interested in and familiar with the characteristics of sparks and flames as the major sources of excitation of metal spectra and their use in conjunction with suitable equipment to serve for atomic absorption spectrometry. Remarkably, the enormous progress in photoelectric detectors in the succeeding decade rendered photographic receivers completely obsolete thereby revolutionizing atomic spectroscopy.

I had been injected into the process of spectroscopy at what seems to have been a virtually ideal time. There had been few changes of profound practical significance in sources of excitation during the preceding one hundred years initiated by Bunsen and Kirchhoff’s studies of flames. The investigation of spectral sources remained an important experimental initiative which could enlarge the scope of spectrochemical analysis.

In particular, the carbon/carbon electrode of high voltage sparks was invented during the Manhattan Project offering new potentials. The data became accessible to experimentation through declassification of that literature. It employs two carbon electrodes, one of which is drilled such as to leave a very thin bottom which becomes permeable to liquid once a spark is struck. This very powerful and controlled spark discharge yields excellent, quantitative, and reproducible data despite variations in sample composition which do not affect the result adversely. The porous cup electrode spark procedure became our standard power source for samples of unknown metal content, and we adopted it widely for that purpose while we investigated and innovated in atomic absorption spectroscopy which had just been recognized.

The tremendous advantages of atomic flame absorption spectroscopy are summarized readily as are the remarkable attributes of this new field of spectrochemistry compared to that prior to its introduction. Its sensitivity is orders of magnitude greater than that of flame atomic emission lowering the limit of detection of most of the elements of biological interest by from three to seven orders of magnitude and is virtually free of any interferences. The accuracy and precision remains at about 1-2% at all levels of metal determination that are of biological interest. There is virtually no matrix interference. The cost of the equipment is relatively low and is mechanically simple. The applicability is broad. Emission equipment of comparably broad gauge utility does not exist, and the lack of simultaneity of analysis for all elements is readily overcome by the flexibility of the instruments that have been developed. The quantitative analysis of metals present in micro or femto molar amounts was suddenly brought to an unprecedented level making the whole field of metal biology accessible.

In view of the relative simplicity of flames, we searched for one that would be much hotter than conventional ones without becoming more complex. The cyanogen/oxygen flame has a temperature of, i.e., 4640°C, i.e., almost double that of the oxygen-hydrogen flame, i.e., 2690°C and we managed to produce and test it. This innovation was greeted warmly by the pros and was a great help in our analytical efforts.

It was a stroke of good fortune that my time at MIT overlapped and coincided with the one truly fundamental step of progress in flame spectroscopy. Flame atomic absorption spectroscopy became the linchpin of our efforts to make biological spectroscopy an intrinsic part of modern biological and medical science.

By 1870 Kirchhoff had clearly established the general laws governing the absorption and emission of energy by atoms, and consequently the principle of atomic absorption. Thus the yellow emission lines originating from sodium in sunlight superimpose exactly on the Fraunhofer D line. In turn, the emission flame of sodium will absorb the same yellow lines emitted when pure sodium serves as the source. Since then, “self-absorption” or “self-reversal” of lines, was recognized to exemplify the principle of atomic absorption that had been observed frequently in emission spectroscopy. In 1955 it was realized that atomic flame absorption results in much more sensitive methods than does atomic emission.

We promptly recognized the importance of this observation to determine the roles and occurrence in minute amounts of metals in biological matter. Hence, we converted our extensive investment in porous cup spark spectroscopy and replaced it by atomic absorption equipment of our own design. As a consequence, we placed ourselves in a strong position to spearhead that field and accomplished just that, as the record shows. This, in turn, revitalized interest in spectrochemistry in general and resulted in the introduction of new generations of equipment, marketing and scientific research in bioinorganic spectroscopy.

All of this coincided with a vast number of other technical and instrumental advances which, of course, greatly furthered progress. New generations of equipment were now brought to markets dedicated to physiology, biochemistry and clinical chemistry. They incorporated advances in optics, flames, electronics and computers.

This remarkable development did not receive as wide recognition in scientific circles as one might have anticipated based on the earlier history of spectroscopy.

These activities and the resultant publications soon earned me recognition as a professional spectrochemist. As a result, I was asked to become a consultant to the Jarrell-Ash Company, the manufacturer of spectrographs and the American collaborator of Adam Hilger Watts Company of London which, at that time, was the most prominent innovator and supplier of spectrochemical equipment, reagents and supplies worldwide. The Humble Oil Refinery of Houston retained me as a consultant with the understanding that I would extend the spectrochemical experience gained in dealing with biological matter to petroleum, fuel oils and other natural products of interest to that company.

We constructed and designed a variety of prototypes which have long since been replaced by completely different models. With some sense of pride, we can point out that in the brief period of five years we succeeded in lowering the limit of detection for transition and IIB metals from micrograms to picograms per ml, remarkable progress, indeed.

This summary of the history of the BRL and CBBSM may be both helpful and instructive to indicate the results of the first ten years of biochemical work accomplished and insights gained based on the considerations described. We rapidly identified a considerable number of zinc enzymes recognized and immensely extended the role which zinc plays in biology.

From 1955 to 1965, the first ten years of the operation of the BRL, the following were identified as zinc metalloproteins or enzymes at the BRL in rapid succession: procarboxypeptidases A, carboxypeptidases A, procarboxypeptidases B, carboxypeptidases B, alcohol dehydrogenases, aldolase, thermolysin, alkaline phosphatase, delta amino levulinic acid dehydratase, RNA polymerases, reverse transcriptases, metallothioneins and alpha₂ macroglobulin.

Since then, literally several thousand zinc metalloenzymes and proteins have been recognized and identified. By now zinc metalloproteins or zinc metalloenzymes are ubiquitous and have become household words. The literature on the subject has long since ceased to be a curio and its ramifications have profoundly altered the biochemical and genetic literature and scene.

There remains the pleasant task to summarize the consequences of the results obtained in the BRL and CBBSM in the course of the last forty years which can justly be attributed to the work performed there.

The wide distribution of zinc, iron and copper and their biological roles in all phyla and species
The pivotal functions of zinc in catalysis by all six known classes of enzymes
The interaction of zinc with enzymes and nucleic acids
The joint participation of zinc with iron, copper and magnesium in catalysis by numerous enzymes
The existence and characteristics of metallothioneins
Alcohol metabolism and daidzin
Angiogenin and Angiogenesis
Developmental biology and cellular differentiation

It is safe to predict that the most far-reaching consequences of these early developments are yet to come.