Thermostability of human serum biotinidase activity.

Each enzyme has its own characteristic thermal stability at the physiological temperature range (20-37 o C) and at a high temperature range (50-60 ’ C). Knowledge of the thermal stability of the enzyme is essential for practical purposes, i.e. manipulation or handling. Thermal stability at the physiological temperature range is essential for studying the effects of endoor exo-type glycosidase or protease on the enzyme activity. Thermostability must also be known at high temperatures for utilizing in biotinidase purification [1,2] or in stopping the enzyme reaction [3]. Purified (21200-fold) human serum biotinidase lost 60% of its original activity after heating at 60 o C for 15 min, as recently reported by Chauhan and Dakshinamurti [4] using Knappe’s calorimetric method. Partially purified hog serum biotinidase (1910-fold) completely lost activity after heating at 60°C for 30 min as reported by Pispa [2]. Bacterial biotinidase of Streptococcu.s fuecafis (strain lOC1) [3] is reportedly heat labile; 92% of the original activity was lost after treatment at 60 o C for 10 min. In this report, we attempted to clarify these conflicting results in a systematic way by a newly developed HPLC biotinidase assay method [5]. We found that biotinidase activity in human native serum was completely inactivated by heat treatment at 60 o C for 15 min. However, simple dilution of the fresh serum with a neutral phosphate buffer or dialysis against the phosphate buffer increased the heat stability of biotinidase activity in fresh serum; 27% and 50% of enzyme activity, respectively, remained after heat treatment at 60 o C for 15 min. Purified human serum biotinidase was relatively heat resistant; 40% of enzyme activity remained after heat treatment at 60 “C for 15 min. This finding is in accordance with the result of Chauhan and Dakshinamurti [4].