Affinity ultrafiltration assay for transferase activity

A method for assaying activity of a transferase comprising the steps of:

• (1) under appropriate conditions which render said transferase active reacting said transferase with a labeled substrate and an unlabeled substrate to yield a product having a label moiety from said labeled substrate and a binding site moiety either from said unlabeled substrate or existing as a specific structure of said product, (2) contacting the reaction mixture with a sufficient amount of a solution of a macroligand having a molecular weight substantially larger than that of said labeled substrate and at least one recognition site specific to said binding site moiety such that at least a substantial fraction of said product forms a complex with said macroligand, (3) subjecting the complex mixture to ultrafiltration with a membrane having a molecular weight cutoff value substantially larger than the molecular weight of said labeled substrate to pass the unreacted labeled substrate but substantially smaller than that of macroligand to retain said complex, (4) washing the retentate containing said complex by said ultrafiltration with an appropriate solution to at least substantially remove the residual of said unreacted labeled substrate from said retentate, and (5) by means of said label moiety determining the final retentate containing the separated product at least substantially free of said unreacted labeled substrate as a measure of said activity of said transferase.