具有cGPX活力的含硒抗体酶的酶学及动力学性质研究

通过单克隆抗体制备技术得到三株特异结合半抗原4(GSH-S-DNP二苄酯)的单克隆抗体HB4,HB5和HB7.抗体经两步化学诱变得到具有细胞谷胱甘肽过氧化物酶(cGPX)活性的含硒抗体酶mHB4,mHB5和mHB7,活力分别为170,1867,32U/μmol.其中mHB5的活力是天然兔肝cGPX的0.32倍,m4A4的1.51倍.等离子体-质谱(ICP/MS)测得每分子含硒抗体酶分子中大约存在2个硒原子.mHB5的最适pH为8.6～8.8.在pH值范围为7.0和37℃条件下,mHB5催化GSH和H₂O₂或t-ROOH反应的二级速率常数为:_k+1(H₂O₂)9.71×10⁶L/(mol·min),_k+1(t-ROOH)5.99×10⁵L/(mol·min).mHB5使非酶催化反应速率提高了9.8×10⁶和3.7×10⁵倍.

Enzymatic and Kinetic Properties of Selenium-containing Catalytic Antibody with cGPX Activity

Three selenium-containing catalytic antibodies mHB4, mHB5 and mHB7, which acted as mimics of cytosolic glutathione peroxidase(cGPX), were prepared by chemically introducing selenium into monoclonal antibodies HB4, HB5 and HB7. HB4, HB5 and HB7 were raised against a GSH derivative GSH S DNP dibenzyl ester. The cGPX activity of mHB4, mHB5, mHB7 were 170, 1 867, 32 U/μmol, respectively. The cGPX activity of mHB5 was 0.32 fold of natural rabbit liver cGPX and 1.51 fold of m4A4. About two atoms of selenium existed in each of mHB5 molecule determined by inductively-coupled plasma/mass spectroscopy(ICP-MS). The optimal activity of mHB5 was at between pH 8.4 and 8.8. The reaction catalyzed by mHB5 involved a Ping Pong mechanism. At pH 7.0 and 37 ℃, the apparent second order rate constants for reaction of mHB5 with H₂O₂ and t ROOH were as followed: _k+1 (H₂O₂)=9.71×10⁶ L/(mol·min), _k+1 ( t ROOH)=5.99×10⁵ L/(mol·min). Rate accelerations( k_cat / K_m/ k_uncat ) 9.8×10⁶ and 3.7×10⁵ fold those of the uncatalytic reaction were observed.