Humic Substances Reduce Inactivation of Alkaline Phosphatase Induced by Ultrasound at Therapeutic Intensities

The present study investigated the influence of ultrasonication and humic substances (HS) on the colloidal-chemical characteristics and enzyme activity of alkaline phosphatase (AP). Specifically, the distribution of AP in immiscible liquid systems, its adsorption at the liquid-liquid interface, and its enzyme activity were examined. The latter was assessed by measuring both the enzyme's ability to hydrolyze 4-nitrophenyl phosphate and its capacity to catalyze the synthesis of calcium phosphate. Under alkaline pH conditions, HS preserved the enzyme activity of AP during ultrasonication. Ultrasonication did not alter the secondary structure of the protein, and the observed loss of enzyme activity is reversible for both free AP and its mixture with HS. This reversibility was further supported by the enzymatic synthesis of calcium phosphate, which yielded comparable results for both treated and untreated AP samples. The following mechanism of HS action in the AP-HS system is proposed: (1) HS fragments form complexes with AP, resulting in higher enzyme activity; (2) continued ultrasonic treatment leads to a sharp decline in the activity of free AP; and (3) after the ultrasonic treatment is completed, the enzyme activity remains reduced; however, complexes between HS fragments and AP may begin to reform and exert their effect again.