Rapid and sensitive determination of protein by light-scattering technique with Eriochrome Blue Black R

Based on the interaction between Eriochrome Blue Black R (EBBR) and proteins, which causes a strong light-scattering signal with the maximum scattering peak located at 398 nm, a simple, rapid, sensitive and selective method is developed for the determination of proteins by the light-scattering technique using a common spectrofluoremeter. Under proper experimental conditions, the protein determination can be performed in the range of 0.1-25, 0.1-20 and 0.25-25 μg ml⁻¹ for bovine serum albumin (BSA), human serum albumin (HSA) and human immunoglobulin G (IgG), respectively. The detection limit, calculated as 3 times the S.D. of nine blank measurements, are 33 μg l⁻¹ for BSA, 25 μg l⁻¹ for HSA and 38 μg l⁻¹ for IgG. Moreover, there is no significant difference among the scattering signals yielded by HSA, IgG and BSA, and almost no interference of many amino acids and metal ions. The method has been satisfactorily applied to the direct determination of the total protein in human serum, saliva and urine samples. The results obtained from the studies on the binding characteristics of EBBR to BSA indicated that an electrostatic force existed in the binding system, and the binding constant (K) and the number of the binding sites (n) at 25 °C are 1.69×10⁵ l mol⁻¹ and 0.946, respectively.