Gupta G S, Sharma P K
Department of Biophysics, Panjab University, Chandigarh, India.
Indian J Biochem Biophys. 1995 Oct;32(5):266-71.
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 (Ea) 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.
干燥状态下质子辐照后睾丸透明质酸酶（HDase）的剂量反应活性曲线遵循复杂机制，可能涉及多次击中及大小可变的多个靶点，从而给出恒定的G值1.66。当将辐照后的酶置于水相时，活性的缓慢恢复似乎会改变靶点分析。然而，1×10⁵至8×10⁵ Gy剂量的辐照模式表明，尽管酶与底物（透明质酸）的结合亲和力增加，表现为Km从500 mg/l降至300 - 70 mg/l，但辐照后的HDase催化反应速率却因反应速度降低而下降（Vmax：8×10⁵ Gy时为266对76单位）。对未辐照的HDase进行热变性的活化分析表明，涉及78 kcal/mol的活化能（Ea），在1×10⁵至8×10⁵ Gy辐照后，残留酶的该值降至63 - 52 kcal/mol。相应的活化熵变（δS）从对照值-291 eu增加至8×10⁵ Gy时的-236 eu。从与水相恢复相关的热力学分析得出结论，HDase因质子能量在包括与分子二级/三级结构相关的氢键在内的弱力之间耗散而失活。
