Effects of Different Molecular Weights of Hyaluronic Acids on Viscosities and Lysozyme- and Peroxidase-related Enzymatic Activities

Abstract

Objectives: Hyaluronic acid has been considered as a candidate molecule for saliva substitutes. To investigate influences of molecular-weight of hyaluronic acid on its rheological and biological properties, we examined viscosities of hyaluronic acids with different molecular-weights and their effects on lysozyme- and peroxidase-related enzymatic activities both in solution and on hydroxyapatite surface. Methods: Four different-sized hyaluronic acids (10 kDa, 100 kDa, 1 MDa, and 2 MDa), hen egg-white lysozyme (HEWL, 30 μg/mL), bovine lactoperoxidase (bLPO, 25 μg/mL), glucose oxidase-mediated peroxidase (GO-PO), and human whole saliva were used. Viscosity values for hyaluronic acids were measured by a cone-and-plate digital viscometer at six different concentrations (0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mg/mL) and six different shear rates (11.3, 22.5, 45.0, 90.0, 225, and 450 s-1). Enzymatic activities of lysozyme, peroxidase, and GO-PO were examined by hydrolysis of fluorescein-labelled Micrococcus lysodeikticus, oxidation of fluorogenic 2,7-dichlorofluorescein (LDCF) to fluorescing 2,7-dichlorofluorescein (DCF), and production of oxidized o-dianisidine, respectively. Lysozyme and peroxidase activity were measured both in solution and on hydroxyapatite surface, and GO-PO activity was examined only in solution. For enzymatic activities, hyaluronic acids at 0.5 mg/mL were used. Results: The 100 kDa-hyaluronic acid at 5 mg/mL, 1 MDa at 0.5 mg/mL, and 2 MDa at 0.2 mg/mL showed similar viscosity values to human whole saliva at the shear rates from 60 to 160 s-1. In solution assays, only 2 MDa-hyaluronic acid inhibited lysozyme activities in saliva significantly. In surface assays, high-molecular-weight hyaluronic acids inhibited lysozyme and peroxidase activities and the inhibitory activities were more apparent on saliva than purified enzymes. GO-PO activities were not significantly affected by all the hyaluronic acids experimented. Conclusions: Hyaluronic acids of low-molecular-weight at high concentrations and high-molecular-weight at low concentrations showed similar viscosity values to human whole saliva, and inhibitory effects of hyaluronic acids on lysozyme and peroxidase activities were more significant in high-molecular-weight ones, on-surface than in-solution, and on saliva than purified enzymes.