Immobilization of the restriction endonucleases PvuII and HindIII

The effects of several chemical reagents on the activity of the restriction endonucleases PvuII and HindIII 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 contains PvuII and PvuII* sites and HindIII and HindIII* sites, did not protect the enzymes from the carbodiimide. On the other hand, glutaraldehyde, which reacts primarily with lysine residues, inactivates PvuII and HindIII enzymes. This specific effect could not be prevented by pBR322 DNA. Preincubation with high concentrations of N-ethylmaleimide, which reacts with sulfhydryl groups, caused slight inhibition of PvuII activity, but had no effect on the activity of HindIII enzyme. The effects of glutaraldehyde, carbodiimide, and N-ethylmaleimide on other restriction endonucleases were also investigated. Restriction endonucleases PvuII and HindIII 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.