Subunits and Allosterism

And then quite by chance I even became involved with "molecular biology,'' the pinnacle of the bright new look of biological research.

After the DNA double helix "molecular biology'' had moved to the top of the league in terms of relevance to the ultimate secrets of life. But proteins remained very much a part of the story. How many polypeptide chains? How big are they? Are they all alike? How are they linked? These questions were fundamental to an understanding of immunoglobulins, as seen in Figure 2, but they were also important for some proteins that were at the core of the genetic mechanism, part of the DNA ! messenger RNA ! protein story, and this made them part of the molecular biology agenda. This circumstance got me involved in a controversy, quite unintentionally; I was not even aware of it at first. The ultimate substantive matter in question was the subunit composition of an enzyme, aspartate transcarbamylase, a critical regulatory component in the biosynthesis of nucleotides - DNA and RNA. But the controversy itself centered on just one person, Jacques Monod, one of the half dozen people closest to the very heart of molecular biology. He shared the Nobel Prize for biology or medicine in 1965 with two of his colleagues from the Pasteur Institute, but has never been one of my favorite scientists. One historian describes him as having a zest for combat and a flair for performance, but I was only rarely impressed by the relevance of his battles to scientific priorities.

The incident to which I am referring here has to do with allosterism, a well-defined concept in the chemistry of some enzymes and in the binding of oxygen to hemoglobin. In Monod's perception it became much more; he saw allosterism as the fundamental element of all biological control; "the most elaborate product of molecular evolution.'' As is well known, allosteric regulation in hemoglobin, is achieved by interaction between protein subunits - the constituent a and ft polypeptide chains. Monod turned this into a generalization: protein sub-units became part of the fundamental principle; although, actually there is no absolute mathematical necessity for multiple subunits to be involved. In any case, subunits and their molecular weights were at the heart of allosterism research and that is how my laboratory came into the picture: in a project dominated by two visiting polymer chemists from overseas, Savo Lapanje from Yugoslavia (now Slovenia) and Kazuo Kawahara from Japan, we had established the potency of concentrated guanidi-nium chloride as a protein denaturant. After disulfide bond disruption, all proteins in that solvent were found to be reduced to their constituent polypeptide chains, behaving physically as structureless "random coils.'' We successively applied a series of discriminating physical methods to show rigorously that under these conditions the molecular behavior became essentially identical to that of a synthetic organic polymer dissolved in an indifferent organic solvent; indeed our project was not primarily directed at protein chemists or biochemists, but rather at polymer chemists, to convince them that it was often valid to apply the theories developed for polymer chemistry to the systems we were working on [25].

Use of concentrated guanidinium chloride as a solvent created a special problem for molecular weight measurements in the ultracentrifuge, where the buoyant mass of the protein particle is the determining factor. This factor includes bound solvent and requires knowledge of the proportions of water to guanidine, within the protein particles as well as in the bulk solvent: is one or the other solvent component preferentially bound? This problem was successfully investigated in my laboratory by Kirby Hade, a postdoctoral fellow who used to be a stockbroker and came to work with me to look for a less volatile career. Characteristically he chose the most esoteric and laborious method for determining preferential binding, by measurement of "isopiestic'' compositions [26] - a method that was exact if you fully understood the partial differential equations that describe what you are doing. George Scatchard was the only chemist who had ever been known to actually use the isopiestic method in a biochemical context. Many years later S.N. Timasheff of Brandeis University became the unchallenged world expert on preferential binding by proteins in multi-component systems - his work should be consulted for detailed analysis [27].

The instigator of the rather acrimonious debate with Jacques Monod was the "molecular biology correspondent'' of the journal Nature, who, at that time, wrote unsigned weekly contributions related to current work in the field. He has since been identified as Walter Gratzer, well known today both as a molecular biologist and as a writer of semipopular works in general science. Gratzer expressed his dismay at the faddishness of the allosteric phenomenon, using the enzyme aspartate transcarbamylase as an example. He noted Monod's enthusiastic "discovery" of the use of guanidine as an agent for denaturation (and polypeptide disruption) and as a medium for measurement of subunit molecular weights. Gratzer pointed out with some sarcasm (perhaps even jokingly) that it should really be called a "rediscovery'' citing the work from my laboratory and others in America as a precedent.

Monod did not take kindly to criticism and attacked Gratzer's judgment in a letter to the journal, which was not a very wise move, considering Monod's own ignorance of physical chemistry. The importance of buoyant density in the interpretation of ultracentrifugal results, apparently just recognized by Monod, was at the core of the dispute. Gratzer appropriately referred to Kirby Hade's earlier analysis of that problem as definitive. Another rather arrogant (as well as puzzling) view of Monod's was that he thought he had free choice in what was a subunit and what was not - aspartate transcarbamylase should be called a tetramer, he asserted, not an octamer, which it seemed to be by actual count. As Gratzer put it, Monod's intention appeared to be "a new definition of a monomer - or monodmer perhaps -elusive, but adaptable.''

Such opportunities for a play on words are rare in serious scientific discourse. Monod did not seem to appreciate the play on words and was offended by being made a laughing stock. As for me, I could claim that work done in my laboratory had actually made an impact on the Olympian heights of molecular biology. (Or, as Gratzer put it in a comment on his own role: "It made me feel that I had not lived entirely in vain.'')

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