Molecular Basis of Biological Stability to High Temperatures.

One of the most plausible explanations for the ability of thermophilic organisms to grow at temperatures that usually are lethal to other organisms is that they possess relatively heat-stable proteins. Substantial support for this hypothesis recently has been furnished by Koffler and his associates,1 2 who showed that cytoplasmic proteins obtained from thermophilic bacteria are strikingly more heatstable than those from mesophiles. Their experiments extend previous reports3-7 that the activity of certain enzymes from thermophiles is more heat-stable than that of similar preparations from mesophilic cells. Unfortunately, the available data, except Campbell's,5 were obtained with impure enzymes and thus do not enable one to distinguish between inherent thermostability and external stabilization. Campbell's experiments, however, were performed with purified enzymes. He showed that crystalline amylase isolated from cultures of Bacillus coagulans grown at 550 C. is markedly more heat-stable than a similar isolate from cultures grown at 350 C. Though one could argue that even crystalline proteins may contain stabilizing contaminants in minute yet effective concentrations, the most reasonable explanation for this finding is that the observed difference in relative thermostability is due to molecular differences and not to external substances. Campbell's observation confirms our own conclusions regarding the relative thermostability of flagella from thermophiles. These experiments were presented in preliminary accounts before2, 8 and are described more fully in this paper. Bacterial flagella are helical organelles made up of fibrous proteins9-15 recently named "flagellins."'1 The characteristic appearance of flagella as observable in the electron microscope can be destroyed by acid,9' 10, 15, 16 heat,2' 8 urea,2, 17 and acetamide.2 Apparently flagella "dissociate" into their component units or multiples thereof; this seems true for the action of acid below pH 349, 14, 15 and is probably true also for the effect of the other agents mentioned. Suspensions of flagella are viscous, but their viscosity decreases markedly as the flagella dissociate. Such a decrease therefore can be used as a measure of stability. The advantage of using flagella as a test system is that highly purified preparations from many different organisms can be made rather easily by the same method involving fractional centrifugation (with or without fractional precipitation with ammonium sulfate). Moreover, because flagella are organized aggregates of protein molecules, they lend themselves to a study of intermolecular as well as intramolecular stability.