Thawing acidity during enzyme

The reaction was carried out in the following manner. Stock blood was transferred into test tubes, water was added, and the solution was frozen and thawed to lyse the cells. At zero time, excess xanthine oxidase was added as a coupling enzyme to convert all the hypoxanthine that was formed during the reaction to uric acid. The reaction was started by the addition of inosine in a phosphate buffer. 

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