The use of ascorbic acid (crystalline vitamin C) as a substrate in oxidase measurements.

During storage the physiological changes in fruits are katabolic, and oxidative processes are doubtless dominant. A study of these processes is of importance from the standpoint of fruit behavior and particularly with reference to physiological diseases such as soft scald and break-down of apples and core break-down of pears. A few degrees difference in storage temperature may mean either severe losses or fruit comparatively free from disease. Thus, at 30^ or 32° F. certain varieties of apples may develop severe soft scald, whereas at 36° this disease very seldom develops. However, at the higher temperature the period of profitable storage is shortened owing to more rapid ripening. These results indicated that a study of the oxidizing enzymes might be of considerable importance for an understanding of the basic causes of this type of disease and in turn might lead to methods for its control. Preliminary tests indicated that the difference in oxidase activity of fruit stored at 32° and 36° might be small. A more sensitive method than had previously been available for measuring small changes in enzyme activity was needed, and the present study was therefore undertaken. Oxidase activity may be determined colorimetrically (16, p. 234)? volumetrically (4), or iodometrically (S). Of these three processes, the last is in many respects the most satisfactory. In Guthrie's (^) iodometric method the oxidizable substrate is prepared by heating glucose with a dilute sodium hydroxide solution. The glucose is broken down into numerous derivatives, some or all of which reduce iodine in acid solution. These carbohydrate degradation products are unstable and lose their oxidizing power in the presence of air, and their oxidation is catalyzed by plant juices, i. e., by oxidase. When glucose is heated with dilute alkali, a mixture of various decomposition products is formed. It is conceivable that some of these carbohydrate derivatives may require different degrees of oxidation before their iodine-reducing power is destroyed, while other products in this mixture may possess no iodine-reducing power or may even have an inhibitory action on the oxidase enzyme. For this reason it would seem desirable to have a pure substrate in which the oxidizable material would be uniform and other products would not be present as a possible source of interference with the enzyme action. In the quantitative determination of ascorbic acid in plant tissues it is necessary to inactivate the plant oxidase to prevent destruction of the acid. Szent-Györgyi {IS) noted the reducing power of plant juices and identified the reducing substance as hexuronic acid. This