Application of the dibromohydroxyphenylfluorone-molybdenum(VI) complex to the sensitive spectrophotometric determination of protein

A new method is described for the spectrophotometric determination of protein, based on the binding interaction of protein, molybdenum(VI) and dibromohydroxyphenylfluorone (DBHPF). At pH 2.0-3.4 and in the presence of Triton X-100, the binding reaction is complete within 8 min at room temperature, and causes a change of the absorption spectrum and an absorbance decrease at 533 nm of the DBHPF-Mo(VI) complex. The calibration graphs for bovine and human serum albumins are both linear up to 8.0 mg l(-1) with 3 sigma. detection limits of 70 and 80 ng ml(-1), respectively. Compared with some accepted and reported assays, besides being highly sensitive, the method is reproducible and simple, and there is a lack of interferences. Results for a culture, cell homogenate and human body fluids are in agreement with those obtained by the Bradford method, with relative standard deviations of 1.5-2.5% (n = 5). The binding numbers and association constants of serum albumins with the complex are estimated by a modified Scatchard graphical method, and the binding mechanism is discussed. (C) 2000 Elsevier Science B.V. All rights reserved.