A Phosphate Staining Reagent Revisited

Abstract

Abstract A phosphate staining reagent [S. K. Goswami and C. F. Frey, J. Liquid Res., 12, 509 (1971)] was found to show differential staining of phosphate esters. Thus, the staining reaction mixtures in cases of molecules like dodecyl dihydrogenphosphate [DDP], phosvitin and phosphocellulose showed intense blue colors which on standing precipitated down leaving a colorless supernatant. However, at equimolar phosphate concentrations molecules like inorganic phosphate/diphosphate, ATP, and phosphoserine stained poorly. It was apparent that (a) the staining does not involve hydrolysis of phosphate esters and (b) the proximity between phosphate esters determines the staining intensity of different molecular systems. In order to confirm the proximity model, constant amounts of DDP were incubated with the staining reagent in the presence of increasing concentrations of sodium dodecyl sulfate [SDS]. A decrease in staining of phosphate was seen with progressive increase in the concentration of SDS. The slope of this inhibition curve was enhanced when the concentration of DDP was clamped at 300 μM (1 M = 1 mol dm−3) than was the case at 450 μM. The results of these experiments are suggestive of competitive binding of SDS to aggregates of DDP, a phenomenon which decreases the proximity relationship between phosphate head groups of DDP. This has provided us a new method for the estimation of SDS and amphiphiles of similar kind which have the ability to alter proximity relations in DDP aggregates. Phosvitin, a phosphoprotein rich in clusters of intramolecular phosphate esters was only marginally sensitive to the presence of SDS for its staining by this reagent.