Determination of protein based on absorbance decrease of molybdenum(VI) complex with 2,6,7-trihydroxy-9-(4′-chlorophenyl)-3H-xanthen-3-one

A new spectrophotometric method is proposed for protein determination with the limit of detection at nanogram levels. At pH 2.2-3.7, and in the presence of Triton X-100, protein, molybdenum(VI) and 2,6,7-trihydroxy-9-(4'-chlorophenyl)-3H-xanthen-3-one (p-Cl-PF) react within 3 min at room temperature to develop a supramolecular complex stable for at least 6 h, resulting in an absorbance decrease at 530 nm of the p-Cl-PF-Mo(VI) complex. The dynamic ranges for bovine and human serum albumins are 0-12 mg l(-1) with detection limits of 30 and 100 ng ml(-1), respectively. The method is simple, practical and relatively free from interference from coexisting substances, as well as much more sensitive than most of the existing assays. The determination results of human body fluid samples are identical to those by the Bradford method, with relative standard deviations of five determinations of 0.7-2.8%. By the Rosenthanl graphic method the binding numbers and association constants of serum albumins with the complex are estimated, and the binding reaction is considered to be mainly due to electrostatic forces.