Immobilization of the restriction endonucleasesPvuII andHindIII

The effects of several chemical reagents on the activity of the restriction endonucleasesPvuII andHindIII were investigated. Carbodiimide, which reacts preferentially with carboxyl groups, was found to inactivate these enzymes. This specific effect could be prevented by Mg²⁺ cation. pBR322 DNA, which containsPvuII andPvuII* sites andHindIII andHindIII* sites, did not protect the enzymes from the carbodiimide. On the other hand, glutaraldehyde, which reacts primarily with lysine residues, inactivatesPvuII andHindIII enzymes. This specific effect could not be prevented by pBR322 DNA. Preincubation with high concentrations ofN-ethylmaleimide, which reacts with sulfhydryl groups, caused slight inhibition ofPvuII activity, but had no effect on the activity ofHindIII enzyme. The effects of glutaraldehyde, carbodiimide, andN-ethylmaleimide on other restriction endonucleases were also investigated. Restriction endonucleasesPvuII andHindIII were immobilized by covalent coupling to various insoluble carriers. Both immobilized enzymes retained partial enzyme activities, when immobilized through phenolic groups and were stable for at least two months.