MICROASSAY FOR THE QUANTITATION OF PROTEIN PRECIPITABLE POLYPHENOLS -USE OF BOVINE SERUM ALBUMIN-BENZIDINE CONJUGATE AS A PROTEIN PROBE

A simple, sensitive and indirect spectrophotometric method for the det ermination of protein precipitable polyphenols (tannins) has been deve loped, based on the ability of the polyphenols to precipitate the synt hetic, brown coloured ate-protein, bovine serum albumin-benzidine conj ugate (BSA-bentidine, mole ratio 1:7), which shows an absorption maxim a at 405 nm. The amount of unprecipitated BSA-benzidine is measured di rectly at 405 nm, which is inversely related to the polyphenol concent ration. Tannic acid was used as a reference standard. The microassay w as performed in citrate/phosphate buffer (0.1 M), pH 4.8. The method w as found to be linear in the range of 5-150 mu g (3-88 nmol) of tannic acid (y = 1.0+(-0.007)x; r = -0.989). Spiking studies carried out wit h various levels of tannic acid (0.01, 0.1 and 1.0%) indicated a recov ery in the range of 94-101% and 94-98% in rice and sorghum samples, re spectively. Free phenolics, when added in the range of 50-150 mu g (ca techin, chlorogenic acid, ferulic acid, caffeic acid and p-coumaric ac id) had no influence on the protein precipitation in the microassay. A lso spectral analysis of free phenolics and acid-methanolic sorghum ex tracts showed no interference in the present method. The conjugate was found to be stable over a period of 24 weeks in a freeze-dried condit ion and at 4 degrees C, with <5% deterioration in aqueous condition. T he microassay method developed has been used for the quantitation of p rotein precipitable polyphenols in various sorghum (Sorghum bicolor L. Moench) genotypes and compared with the widely used Folin-Denis chemi cal method of analysis. (C) 1998 Elsevier Science Ltd. All rights rese rved.