The determination of carbon dioxide in fermenting mixtures. Co-zymase: a study of purification methods

摘要

THE DETERMINATION OF CARBON DIOXIDE IN FERMENTING MIXTURES.ududIn connection with a research on enzymic behavior it was necessary to develop a simple and expeditious method for the determination of the carbon dioxide formed during fermentations. The most suitable method of carbon dioxide analysis appeared to be the procedure evolved by Cain and Maxwell for the determination of carbon in steel. They absorbed the carbon dioxide, formed by combustion, in known volumes of barium hydroxide solution, and followed the precipitation of barium carbonate by measurements of the electrical conductivity of the solution. The idea has been applied by Spoehr and McGee to the determination of carbon dioxide in their studies on plant respiration. This paper describes the development of this general method so as to render it applicable to studies on fermentation.ududThe technique which was finally adopted consisted in liberating the carbon dioxide from the fermentation mixtures by shaking, carrying it by means of stream of air, free from carbon dioxide to the absorption vessels containing barium hydroxide, and measuring the change in resistance of the barium hydroxide solutions.ududCO-ZYMASE: A STUDY OF PURIFICATION METHODS.ududCo-zymase is one of the essential components of the complex enzyme mixture which effects alcoholic fermentation in the absence of living cells. The separation of the mixture into "zymase" and "co-zymase" was first accomplished by Harden and Young by means of ultrafiltration through a gelatin-impregnated Chamberland filter candle. The residue and filtrate as thus prepared possessed, separately, no fermentative action, but when mixed were found to produce a rapid fermentation. The active constituent of the residue was names zymase, while that constituent of the filtrate responsible for the reactivation of the residue was named co-zymase.ududThe mechanism of the activation of the zymase was investiaged by Euler and Myrbach, who came to the conclusion that the co-zymase was involved in that stage of the process in which inorganic phosphates are converted to carbohydrate esters. The same authors have described a comprehensive and successful series of experiments of the purification of co-zmase.ududA complete knowledge of the properties and function of co-zymase would be of extreme importance, as it should throw considerable light upon the mechanism of enzyme action as a whole, and should in addition have important bearing upon the entire question of carbohydrate utilization. It has been shown, for example, by Myerhof that a substance, either identical with, or very similar to co-zymase exists in most animal tissue, and the demonstration of a complete identity of the two would have important metabolic connotations.ududIn view of the importance of the field, as well as of the interest of one of us in the problem of carbohydrate utilization, we decided to attempt an extension of the work of Euler and Myrbach on the purification of co-zymase. At the beginning, however, we found that our yeast produced much less active enzyme preparations that those of Euler and Myrbach, as regards both the zymase and co-zymase content, and also that the technic of the above authors with lead precipitation, which enabled them to secure an initial purification of from ten to thirty fold, was in our case practically useless.ududWe therefore abandoned our original intention and have instead studied the purification produced in our material by a variety of reagents. In the investigation we have repeated much of the work done by Euler and Myrbach, and several differences have been found, which appear difficult to explain solely upon the basis of the lower initial purity of our material. As certain of the experiments show distinct promise, we hope to be able to extend the work upon a material of considerably higher original purity, such as was employed by Euler and Myrbach.