IMMOBILIZED NITRO-AVIDIN AND NITRO-STREPTAVIDIN AS REUSABLE AFFINITY MATRICES FOR APPLICATION IN AVIDIN-BIOTIN TECHNOLOGY

Chemically modified forms of egg-white avidin and bacterial streptavid in (termed nitro-avidin and nitro-streptavidin, respectively), in whic h the binding-site tyrosine was nitrated, were used for several biotec hnological applications. The fundamental difference between nitro-avid in and the native protein is that interaction of the modified protein with biotin can be reversed under relatively mild conditions. Conseque ntly, nitro-avidin affinity columns or immobilizing matrices can be re used. Three examples are given to demonstrate the possible uses of suc h columns: (a) biotinylated protein A was attached to a nitro-avidin a ffinity column, and immunoglobulin was purified directly from whole ra bbit serum; (b) biotinylated transferrin was attached to a nitro-strep tavidin column, and anti-transferrin was isolated directly from rabbit anti-serum; and (c) biotinylated beta-glucosidase was immobilized ont o a nitro-avidin column and used as an enzyme reactor. In each example , the immobilized biotinylated probe could be released selectively fro m the column and recovered following its utilization. Reusable nitro-a vidin thus provides an easy and attractive reversible form of avidin a nd thereby serves to expand the versatility of avidin-biotin technolog y. (C) 1996 Academic Press, Inc.