title: Molecular inactivation of testicular hyaluronidase in solid state after proton irradiation: a study based on target size, substrate binding and thermodynamic analysis of heat denaturation.
abstract: Dose response activity curve of testicular hyaluronidase (HDase) following proton irradiation in dry state follows complicated mechanisms which may involve multiple hits and multiple targets of variable sizes giving a constant G value of 1.66. Target analysis appears to be modified by slow recovery of activity when irradiated enzyme is brought to aqueous phase. However, pattern of irradiation at a dose of 1 x 10(5) to 8 x 10(5) Gy reveals that though binding affinity of enzyme to the substrate (hyaluronic acid) increases as shown by declining Km from 500 mg/l to 300-70 mg/l, the reaction rate of catalysis by irradiated HDase is decreased due to decrease in reaction velocity (Vmax: 266 versus 76 units at 8 x 10(5) Gy). Activation analysis of heat denaturation of nonirradiated HDase suggested the involvement of 78 kcal/mole of energy of activation (E*a) which declined to 63-52 k cal/mole after irradiation at 1 x 10(5) to 8 x 10(5) Gy for residual enzyme. The corresponding change in entropy of activation (delta S*) increased from a control value of -291 eu to -236 eu at 8 x 10(5) Gy. From thermodynamic analysis in association with recovery in aqueous phase, it is concluded that HDase is inactivated due to dissipation of proton energy among weak forces including H bonds associated with secondary/tertiary structure of molecules.
authors.affiliation: Department of Biophysics, Panjab University, Chandigarh, India.
authors.forename: G S
authors.initials: GS
authors.lastname: Gupta
authors.forename: P K
authors.initials: PK
authors.lastname: Sharma
mesh_terms: D000818:Animals; D006358:Hot Temperature; D006821:Hyaluronoglucosaminidase / Q000528:radiation effects*; D008297:Male; D011489:Protein Denaturation; D011522:Protons; D012756:Sheep; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*; D013816:Thermodynamics
chemical_list: D011522:Protons; D006821:Hyaluronoglucosaminidase