Colorimetric Determination of the Purity of 3′-Phospho Adenosine 5′-Phosphosulfate and Natural Abundance of 3′-Phospho Adenosine 5′-Phosphate at Picomole Quantities

Abstract

This work presents novel colorimetric methods not only to measure 3′-phospho adenosine 5′-phosphate (PAP) and 3′-phospho adenosine 5′-phosphosulfate (PAPS) in the range of picomoles, but also to determine the purity of PAPS or PAP contaminants in PAPS in the range of nanomoles. These methods exploit the availability of overexpressed phenol sulfotransferase (PST) and the fact that sulfuryl group transfer requires the use of PAP or PAPS as a cofactor or cosubstrate. Experimental results indicate that absorption at 400 nm due to the production of 4-nitrophenol (pNP) is catalyzed by PST when the sulfuryl group transfers from 4-nitrophenylsulfate (pNPS) to PAP or to 2-napthol. In the absence of an acceptor substrate, PAPS is hydrolyzed to PAP by PST and is determined by sulfation with pNPS before and after this reaction. The change of absorption of pNP at 400 nm corresponds to the amount of PAP that is hydrolyzed from PAPS. Moreover, a standard curve is constructed using authentic PAP and PAP-free PST. Furthermore, this curve is used to determine the amount of PAP in extracts of pig liver, rat liver, andEscherichia coli.