Affinity purification of newly phosphorylated protein molecules. Thiophosphorylation and recovery of histones H1, H2B, and H3 and the high mobility group protein HMG-1 using adenosine 5'-O-(3-thiotriphosphate) and cyclic AMP-dependent protein kinase.

Methods are described for the thiophosphorylation and recovery of histones and non-histone proteins modified by kinases in uitro and in isolated cell nuclei.The ATP analogue adenosine 5'-0-(3-thiotriphosphate) is shown to be an effective donor of thiophosphoryl groups in the reaction catalyzed by the cyclic AMPdependent protein kinase of calf thymus lymphocytes.Substrates for the in uitro reaction include histones HI, H2B, and H3 and the high mobility group protein HMG-1.The modification of these proteins by thiophosphorylation permits separation of the newly modified molecules by affinity chromatography on organomercurial-Sepharose columns.In the case of proteins which lack sulfhydryl groups, such as calf thymus histone H1, separation of previously phosphorylated and nonphosphorylated forms from thiophosphorylated molecules is achieved directly.For proteins containing -sH groups, such as histone H3, treatment with iodoacetate at pH 8 is used to block the cysteine "SH groups selectively before affinity chromatographic separation of the thiophosphorylated molecules.Alternatively, histone H3 can be treated with iodoacetate before enzymatic thiophosphorylation and affinity chromatography.Incubation of isolated Physarum nuclei with [35S]thiophosphate results in labeling of endogenous H1 molecules which can be extracted and separated by organomercurial-Sepharose chromatography.