A PHOSPHATE STAINING REAGENT REVISITED

A phosphate staining reagent [S. K. Goswami and C. F. Frey, J. Liquid Res., 12, 509 (1971)] was found to show differential staining of phosp hate esters. Thus, the staining reaction mixtures in cases of molecule s like dodecyl dihydrogenphosphate [DDP], phosvitin and phosphocellulo se showed intense blue colors which on standing precipitated down leav ing a colorless supernatant. However, at equimolar phosphate concentra tions molecules like inorganic phosphate/diphosphate, ATP, and phospho serine stained poorly. It was apparent that (a) the staining does not involve hydrolysis of phosphate esters and (b) the proximity between p hosphate esters determines the staining intensity of different molecul ar systems. In order to confirm the proximity model, constant amounts of DDP were incubated with the staining reagent in the presence of inc reasing concentrations of sodium dodecyl sulfate [SDS]. A decrease in staining of phosphate was seen with progressive increase in the concen tration of SDS. The slope of this inhibition curve was enhanced when t he concentration of DDP was clamped at 300 muM (1 M=1 mol dm-3) than w as the case at 450 muM. The results of these experiments are suggestiv e of competitive binding of SDS to aggregates of DDP, a phenomenon whi ch decreases the proximity relationship between phosphate head groups of DDP. This has provided us a new method for the estimation of SDS an d amphiphiles of similar kind which have the ability to alter proximit y relations in DDP aggregates. Phosvitin, a phosphoprotein rich in clu sters of intramolecular phosphate esters was only marginally sensitive to the presence of SDS for its staining by this reagent.