Elucidation of the quaternary structure of reversibly immobilized alkaline phosphatase derivatives.

Escherichia coli alkaline phosphatase has been reversibly immobilized on Sepharose CL-4B through two different methods, both based on a disulfide linkage, under conditions selected to favour the coupling of the enzyme to the solid support through one covalent linkage. The quaternary structure of the reversibly immobilized subunit, produced by dissociation of the matrix-bound dimer, was examined by cross-linking with the bifunctional reagent dimethyl suberimidate. Following release from the solid support, the protein was analysed by sodium dodecyl sulfate gel electrophoresis demonstrating the presence of a sufficient amount of dimeric structures in the immobilized subunit preparation to account for all the enzyme activity observed in this sample. These results suggest that the subunit of alkaline phosphatase may be catalytically inactive. This approach to studying the quaternary structure of immobilized subunit derivatives offers the opportunity to directly determine the homogeneity and structure of matrix-bound 'monomer' preparations and is particularly useful in determining if low levels of catalytic activity observed in some immobilized subunit populations are due to the presence of contaminating oligomeric structures.