Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis

Abstract

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 ${\mathrm{PO}}_3^{-}$ monomers with toluidine blue or 4',6-diamidino-2-phenylindole. PolyP chain lengths longer than 62 ${\mathrm{PO}}_3^{-}$ monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich "Type 45", estimated mean length of 45 ${\mathrm{PO}}_3^{-}$ monomers), trimetaphosphate (trimetaP: 3 ${\mathrm{PO}}_3^{-}$ ring), tripolyphosphate (tripolyP), pyrophosphate (PP_i ), and inorganic orthophosphate (o-P_i ) were visualized after separation by an in situ hydrolytic degradation process to o-P_i that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol-chloroform extraction was confirmed with the Friedman test. ³¹ P diffusion-ordered NMR spectroscopy confirmed that extraction also reduced PP_i diffusivity by <10%. Linear regression between the R_f peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 ${\mathrm{PO}}_3^{-}$ monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-P_i , but no PP_i .

Keywords: 31P DOSY NMR; PAGE; Saccharomyces cerevisiae; migration; polyphosphates.