A novel method for the determination of protein utilizing 4-azochromotropic acid phenylfluorone–molybdenum(VI) complex

Abstract In chloroacetate buffer medium at pH 2.0–3.6, and in the presence of p -octylpolyeneglycol phenylether, protein binds 4-azochromotropic acid phenylfluorone–molybdenum (VI) complex rapidly (within 10 min at room temperature) to form a product stable for 3.5 h, leading to the change of the absorption spectrum of the complex and the decrease in the absorbance at 534 nm. Based on this, a sensitive method for the determination micro amounts of protein was proposed. The calibration curve for human serum albumin is linear over the range 0–20 mg l −1 with a detection limit of 0.85 mg l −1 . Very few coexisting substances interfere with the assay. The method was applied to determinate the total protein in biochemical materials with the same results as by the Bradford method. The maximum binding numbers of albumins with the complex were measured by the molar ratio method.