Validity of Iodine and Copper Reduction Methods for Amylase

Abstract

Summary

The achromic iodine method, with several variations, has been investigated for its validity in determining amylase.

The iodine method is not a valid quantitative procedure for the estimation of amylase. There are considerable differences in the quantities of enzyme required to digest different samples of soluble starch to the same end-point in the same time, under identical experimental conditions.

The copper reduction method is a valid quantitative method for measuring the saccharogenic power of an amylase. Dextrin and 3 samples of soluble starch were found to be saccharified at the same rate.

Apparently the disparities in digestibility which exist among different soluble starch samples are caused by varying relative amounts of amylopectin, amylose and dextrins.

If a quantitative method for the determination of amylase be valid and practical, different samples of the substrate—soluble starch—must give similar and reproducible results.

In a previous paper 1 the Wohlgemuth and viscometric methods were investigated by comparing the amounts of enzyme required to produce a given result in different samples of soluble starch. Both of these methods measures the dextrinogenic constituent of amylase; the Wohlgemuth was found to be non-valid as a quantitative procedure, while the viscometric method seems to be valid if properly controlled. Thompson, McGarvey and Wies 2 have obtained good results with the viscometric method by mixing together different samples of soluble starch.

The purpose of the present paper is to investigate the iodine and copper reduction methods as indicated above; the latter method is for the measurement of the saccharogenic agent of the amylase complex.

A. The achromic iodine method and variations.

The first procedure used was that devised by Johnson. 3 The determinations were conducted exactly as specified in his paper; no sodium chloride or phosphate buffer were used. The substrate was a 2% soluble starch preparation; correction was made for the moisture content.

Into each of a series of flasks in a water bath at 40°C. were placed 50 ml. portions of the soluble starch, which was allowed to come to the temperature of the bath. At 10 second intervals, successively increasing amounts of dilute saliva were added to the flasks. (Different specimens of human saliva diluted with different volumes of distilled water were used.) At the end of 10 minutes, 5 drops were removed from the digest and added to 5 ml. of N/8000 iodine solution. The first tube in which no color was developed was taken as the end-point. The quantity of enzyme solution which would just digest the starch to the end-point was determined as accurately as was practicable. The figures given in Table I under “ml. enzyme solution” represent the quantity of dilute saliva required to hydrolyze the soluble starch to the end-point in 10 minutes.