Wondrak E M, Copeland T D, Louis J M, Oroszlan S
Laboratory of Molecular Virology and Carcinogenesis, NCI-Frederick Cancer Research Facility, Maryland 21701.
Anal Biochem. 1990 Jul;188(1):82-5. doi: 10.1016/0003-2697(90)90530-m.
A solid phase assay for human immunodeficiency virus (HIV) protease using an immobilized substrate, Affi Gel 10-Gly-Gly-Gly-Gly-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[3H]Gly-OH has been devised. The Tyr-Pro bond of the substrate was hydrolyzed by the protease, releasing the radiolabeled cleavage product, Pro-Ile-Val-Gln-[3H]Gly-OH, into the supernatant. The pH optimum was found to be 6.0, and a high ionic strength was required for maximal activity. The solid phase assay is usable for convenient monitoring of purification procedures, and rapid screening of inhibitors of HIV protease.
已设计出一种使用固定化底物Affi Gel 10-甘氨酰-甘氨酰-甘氨酰-甘氨酰-缬氨酰-丝氨酰-谷氨酰胺-天冬酰胺-酪氨酰-脯氨酰-异亮氨酰-缬氨酰-谷氨酰胺-[³H]甘氨酸-OH进行人免疫缺陷病毒（HIV）蛋白酶的固相测定法。底物的酪氨酰-脯氨酰键被蛋白酶水解，将放射性标记的裂解产物脯氨酰-异亮氨酰-缬氨酰-谷氨酰胺-[³H]甘氨酸-OH释放到上清液中。发现最适pH为6.0，最大活性需要高离子强度。该固相测定法可用于方便地监测纯化过程以及快速筛选HIV蛋白酶抑制剂。
