Chemical Tools for Study of Phosphohistidine: Generation of Selective Τ-Phosphohistidine and Π-Phosphohistidine Antibodies

Non-hydrolysablestable analogues of τ-pHis and π-pHis have been designed using electrostaticsurface potential calculations, and subsequently synthesized. The τ-pHis andπ-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13,respectively) were used as haptens to generate pHis polyclonalantibodies. Both τ-pHis and π-pHis conjugates in theform of a BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis weresynthesized and characterized by 31P NMR spectroscopy. Commerciallyavailable τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies wereused to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in acompetitive ELISA. Subsequently, the selectivity of the generated pHisantibodies generated using phosphopyrazole 8 and pyridyl amino amide 13as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr,τ-pHis and π-pHis. Antibodies generated using the phosphopyrazole 8 as ahapten were found to be selective for τ-pHis, and antibodies generated usingthe pyridyl amino amide 13 were found to beselective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA,western blot, and immunofluorescence. The τ-pHis antibody was also shown to beuseful in the immunoprecipitation of proteins containing pHis