Husain S, Husain Q, Saleemuddin M
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, India.
Indian J Biochem Biophys. 1992 Dec;29(6):482-6.
Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.
为了了解固定化方法的性质对这些过程的影响，研究了通过将氨基酸侧链氨基或碳水化合物穗与基质偶联而固定化的辣根过氧化物酶（HRP）的耐热性、尿素诱导的失活以及变性后恢复活性的能力。制备了各种固定化制剂并研究了它们的性质：Sp-HRP是通过将HRP直接偶联到溴化氰活化的琼脂糖上获得的，Sp-NHHRP是通过将高碘酸盐氧化和二胺处理的酶偶联到溴化氰活化的琼脂糖上获得的，SpNH-COHRP是通过将高碘酸盐处理的酶偶联到氨基琼脂糖上获得的，SpCon A-HRP是通过将酶结合到Con A-琼脂糖上获得的。与天然HRP相比，所有固定化制剂对热诱导失活均表现出更高的稳定性。Sp-NHHRP最稳定，其次是Sp-HRP、SpNH-COHRP和SpCon A-HRP。Sp-NHHRP在热变性后恢复酶活性的能力也更优，尽管Sp-HRP在尿素变性后恢复的活性最大。对于所有制剂在尿素变性后的再活化，加入Ca2+是必不可少的。
