PROCEDURE FOR COVALENTLY IMMOBILIZING ENZYMES WHICH PERMITS SUBSEQUENT RELEASE

A new procedure for immobilization of proteins through covalent attachment to glass surfaces via thio-ester-containing coupling chains has been achieved, Porous succinamidopropyl-glass beads were converted into the acyl chloride derivative by using anhydrous thionyl chloride and then treated with either 3-mercaptopropionic acid or mer-captoacetic acid. Thus derivatized, the beads were dried in vacuo; they could then be stored for at least 6 months without detectable loss of reactivity with proteins. Immobilization of protein can be readily achieved simply by suspending the derivatized beads in buffer (pH 5-8) and recycling the protein solution through the beads. In studies with a-chymotrypsin, 10-20 mg/g could be conveniently immobilized by this procedure. Experiments with model compounds revealed that immobilization to such derivatized beads occurred by reaction with amino groups. Following immobilization, protein molecules can be released from the surface by cleavage of the coupling chains at the thioester linkage through treatment with 1.0 M hydroxylamine at pH 7.0. Protein thus released contains a free sulfhydryl group at each site of previous attachment, which can be analyzed quantitatively. Application of the immobilization and release procedures to chymotrypsin permitted direct assessment of the kinetic effects of pore diffusional inhibition on the immobilized enzyme preparations and revealed no inherent alteration of the enzyme's kinetic behavior as a result of the chemical derivatization per se. © 1979, American Chemical Society. All rights reserved.