Glaucoma Service, Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.
J Glaucoma. 1994 Spring;3(1):5-11.
Accurate determination of protein concentration in the aqueous humor is important in the interpretation of experiments relevant to the pathogenesis and therapy of glaucoma. We made preliminary observations that the high concentration of ascorbic acid in aqueous humor might interfere with commonly used protein determination assays. Therefore, we tested the effect of ascorbic acid upon absorbance in the bicinchoninic acid (BCA), Lowry, and Bradford assays. Ascorbic acid and bovine serum albumin, alone and in combination, were added to BCA, Lowry, and Bradford reagents in 96-well plates. Absorbance was measured at 562 nm (BCA) and 590 nm (Lowry and Bradford). As little as 40 ng and 28 mug of ascorbic acid (typical aqueous humor concentration is 200-400 mug/ml) altered absorbance in the BCA and Lowry assays, respectively. The Bradford assay was not affected by ascorbic acid. Because of the high ascorbic acid and low protein concentration in aqueous humor, assays such as the BCA and Lowry, which are dependent upon oxidation-reduction reactions, must be used with caution. In most situations, the Bradford method is best for determination of aqueous humor protein concentration.
准确测定房水中的蛋白质浓度对于解释与青光眼发病机制和治疗相关的实验非常重要。我们初步观察到，房水中高浓度的抗坏血酸可能会干扰常用的蛋白质测定方法。因此，我们测试了抗坏血酸对双缩脲（BCA）、Lowry 和 Bradford 测定法中吸光度的影响。将抗坏血酸和牛血清白蛋白单独和混合添加到 96 孔板中的 BCA、Lowry 和 Bradford 试剂中。在 562nm（BCA）和 590nm（Lowry 和 Bradford）处测量吸光度。仅 40ng 和 28µg 的抗坏血酸（典型的房水浓度为 200-400µg/ml）就分别改变了 BCA 和 Lowry 测定法中的吸光度。Bradford 测定法不受抗坏血酸影响。由于房水中的抗坏血酸含量高且蛋白质浓度低，因此必须谨慎使用依赖氧化还原反应的测定法，如 BCA 和 Lowry。在大多数情况下，Bradford 法最适合测定房水中的蛋白质浓度。
