Mattoo R L, Ishaq M, Saleemuddin M
Department of Clinical Biochemistry, Sher-i-Kashmir Institute of Medical Sciences, Kashmir, India.
Anal Biochem. 1987 Jun;163(2):376-84. doi: 10.1016/0003-2697(87)90238-7.
Protein estimation in crude homogenates of plant tissues rich in phenols and phenolases was carried out by the dye-binding and, with recommended cautions, by the Lowry et al. methods and the two were compared. The dye-binding method gave grossly erroneous results with a high degree of variation when the homogenizing media differed; this was not due either to the interference by the components of the homogenizing media or to any shift in the absorbance maximum. While the reduced form of the "derived" polyphenolic compounds, generated during tissue homogenization, appeared to enhance dye binding with bovine serum albumin, their influence on the protein assay directly in crude homogenates was extremely diverse. Tissue homogenization in the absence of a reducing agent results in polyquinone-protein complexes which prevent optimal dye binding, resulting in low protein values, while the endogenous phenolics in a homogenate prepared in a mixture of cysteine and NaCl appear to suppress dye-protein complex formation. It is therefore our opinion that the dye-binding method is unsuitable for protein assay in phenol- and phenolase-rich plant tissues.
采用染料结合法，并在采取推荐的预防措施的情况下，运用洛瑞等人的方法，对富含酚类和酚酶的植物组织粗匀浆中的蛋白质进行了测定，并对这两种方法进行了比较。当匀浆介质不同时，染料结合法给出的结果存在严重误差且变化程度很高；这既不是由于匀浆介质成分的干扰，也不是由于最大吸光度的任何偏移。虽然在组织匀浆过程中产生的“衍生”多酚类化合物的还原形式似乎增强了与牛血清白蛋白的染料结合，但它们对粗匀浆中蛋白质测定的直接影响却极为多样。在没有还原剂的情况下进行组织匀浆会导致聚醌 - 蛋白质复合物的形成，从而阻止最佳的染料结合，导致蛋白质值偏低，而在半胱氨酸和氯化钠混合物中制备的匀浆中的内源性酚类物质似乎抑制了染料 - 蛋白质复合物的形成。因此，我们认为染料结合法不适用于富含酚类和酚酶的植物组织中的蛋白质测定。
