Morrissey T B, Woltering E A
Department of Surgery, Oregon Health Science University, Portland 97201.
J Surg Res. 1989 Sep;47(3):273-5. doi: 10.1016/0022-4804(89)90120-0.
We have previously noted that high levels (10 mM) of calcium interfere with protein determination in the Lowry assay. While there are numerous reports in the literature of substances interfering with the Lowry assay, none of these interfering substances are as ubiquitous as calcium. We hypothesized that physiologic levels of calcium may interfere with the protein determination as measured by the Lowry assay. To test this hypothesis, bovine serum albumin standards from 12.5 to 1600 micrograms/ml were tested in the Lowry assay in the presence of calcium chloride concentrations from 0 to 5 mM. In low protein concentrations (12.5-50 micrograms/ml) 1 to 2 mM calcium induced errors of up to 384%. In samples containing 100 to 400 micrograms/ml protein, 1 to 2 mM calcium induced errors of up to 40%. The addition of sodium oxalate to calcium-spiked protein samples significantly reduced the errors in protein determination at all calcium concentrations tested. This effect also is seen throughout the range of protein concentrations tested (50-1600 micrograms/ml). In most cases sodium oxalate pretreatment of samples used in the Lowry assay reduces errors by 70-95%. Physiologic levels of calcium induce significant errors in the estimation of protein concentration using the Lowry method. Routine pretreatment of Lowry samples with sodium oxalate will provide a more accurate estimation of the protein concentration in biological systems.
我们之前已经注意到，高浓度（10 mM）的钙会干扰洛瑞法中的蛋白质测定。虽然文献中有许多关于干扰洛瑞法的物质的报道，但这些干扰物质都不如钙那么普遍存在。我们推测生理水平的钙可能会干扰洛瑞法测定的蛋白质含量。为了验证这一假设，我们在氯化钙浓度为0至5 mM的情况下，用洛瑞法检测了浓度为12.5至1600微克/毫升的牛血清白蛋白标准品。在低蛋白浓度（12.5 - 50微克/毫升）下，1至2 mM的钙会导致高达384%的误差。在含有100至400微克/毫升蛋白质的样品中，1至2 mM的钙会导致高达40%的误差。向添加了钙的蛋白质样品中加入草酸钠，在所有测试的钙浓度下，都能显著降低蛋白质测定中的误差。在整个测试的蛋白质浓度范围（50 - 1600微克/毫升）内都能看到这种效果。在大多数情况下，用草酸钠对用于洛瑞法的样品进行预处理可将误差降低70 - 95%。生理水平的钙会在使用洛瑞法估计蛋白质浓度时导致显著误差。用草酸钠对洛瑞法样品进行常规预处理将能更准确地估计生物系统中的蛋白质浓度。
