Patel Samir P, Patel Minal A, Modi Hiren R, Katyare Surendra S
Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat 390 002, India.
Indian J Biochem Biophys. 2007 Apr;44(2):88-93.
The conventional method of Fiske and Subba Row for the estimation of inorganic phosphate (Pi) is although rapid, but suffers from the disadvantage that the color is unstable and hence the optical density (OD) measurements have to be carried out within a short time span of 8-12 min. This poses a restriction on the number of samples, which can be handled in a batch. Although, modified procedures involving use of alternate reducing agents/or increasing the concentration of H2SO4 in conventional method have been subsequently developed, but the problem of color stability could not be solved. In addition, the use of higher concentrations H2SO4 has rendered the methods unsuitable in enzyme assays, especially if the acid labile phosphate containing substrates have been used. In the present study, attempts have been made to suitably modify the method to improve the stability of the color and sensitivity and also for its applicability in enzyme assays, especially when acid labile phosphate containing substrates such as ATP is used. We used the higher concentrations (0.625, 0.8 and 1.0 N) of H2SO4 rather than 0.5 N used in the conventional assay procedures. Under these conditions, the reagent blanks do not develop color for up to 24 h, whereas the intensity of the molybdenum blue color in the standard and/or experimental tubes increased with time reaching optimum value at 24 h. Simultaneously, the absorption maximum shifts from 660 nm to 820 nm. The highest concentration of H2SO4 (1.0 N) is found to be the most effective in the process of color development. The sensitivity of the method is from 1.7 to 2.1 times higher, as compared to the conventional Fiske and Subba Row method for the measurements carried out at the end of 15 min at 820 nm and with the highest concentration of H2SO4 (1.0 N); the sensitivity increased 4.8-fold at the end of 24 h. Presence of glucose and sucrose (1-10 mM), NaCl and KCI (5-100 mM), MgCl2 (1-10 mM) and BSA (10 to 500 microg per assay tube) do not interfere either with color development or with OD measurements. The extent of ATP hydrolysis is 1.6 to 3.4% for up to 1 hi, depending upon the concentration of H2SO4 used. Only negligible hydrolysis of G6P is observed under these conditions. These results suggest that the presently modified method is suitable for Pi analysis in the enzyme assays, in the presence of labile phosphate containing substrates.
Fiske和Subba Row用于估算无机磷酸盐（Pi）的传统方法虽然快速，但存在颜色不稳定的缺点，因此必须在8 - 12分钟的短时间内进行光密度（OD）测量。这对一批能够处理的样品数量构成了限制。尽管随后开发了涉及使用替代还原剂/或提高传统方法中硫酸浓度的改进程序，但颜色稳定性问题仍未解决。此外，使用较高浓度的硫酸使得这些方法不适用于酶测定，特别是如果使用了含酸不稳定磷酸盐的底物。在本研究中，已尝试对该方法进行适当修改，以提高颜色稳定性和灵敏度，并使其适用于酶测定，特别是在使用含酸不稳定磷酸盐的底物如ATP时。我们使用了比传统测定程序中使用的0.5 N更高浓度（0.625、0.8和1.0 N）的硫酸。在这些条件下，试剂空白在长达24小时内不显色，而标准管和/或实验管中钼蓝颜色的强度随时间增加，在24小时达到最佳值。同时，最大吸收波长从660 nm移至820 nm。发现最高浓度的硫酸（1.0 N）在显色过程中最有效。与传统的Fiske和Subba Row方法相比，在820 nm处15分钟结束时进行测量且使用最高浓度的硫酸（1.0 N）时，该方法的灵敏度高1.7至2.1倍；在24小时结束时灵敏度提高了4.8倍。葡萄糖和蔗糖（1 - 10 mM）、NaCl和KCl（5 - 100 mM）、MgCl2（1 - 10 mM）以及BSA（每个测定管10至500微克）的存在既不干扰显色也不干扰OD测量。根据所使用的硫酸浓度，高达1小时内ATP的水解程度为1.6%至3.4%。在这些条件下，仅观察到G6P的水解可忽略不计。这些结果表明，目前改进的方法适用于在含不稳定磷酸盐底物存在下的酶测定中的Pi分析。
