A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays

A simplified method for inorganic phosphate determination has been developed. The method is sensitive, easy, economic, and applicable for estimation of phosphate released in both enzymatic and nonenzymatic reactions. A mixture of hydrazine sulfate and ascorbic acid was used as the reducing agent and the conditions for the development of the molybdenum blue color were optimized. Thus in the 4.0 ml assay system, 0.4 ml of the reducing agent solution containing 20 mg each of hydrazine sulfate and ascorbic acid per milliliter of 1.0 N H2SO4 gave a rapid optimum color development with absorption maximum at 820 nm. Color development showed a linear relationship up to 10 microg Pi concentration. Thus the method has a 2.5x higher range of Pi estimation than that of the Bartlett method. The molar extinction coefficient at 820 nm was higher than that obtained in the Bartlett procedure. Also the molybdenum blue color formed was stable up to 24 h. Under the standard assay conditions, interference from acid-labile phosphate as in the case of Na+,K+ ATPase was at the minimum. The applicability of the method for assay of microsomal Na+,K+ ATPase and glucose-6-phosphatase was checked in microassays (final volume 0.1 ml) in comparison to the conventional procedures which use 3-4 times higher volumes. Likewise the applicability of the method for phospholipid analysis was compared with that of the conventional Bartlett method. Under both test systems the results obtained by the micromethod were identical to those obtained by the conventional methods. In general the method, which rapidly produces quantitatively molybdenum blue color, not only is rapid economical, and convenient but also has wide applicability.     

A new enzymatic method for the determination of inorganic phosphate and its application to the nucleoside diphosphatase assay

A new enzymatic method has been developed for the determination of inorganic phosphate, in which purine nucleoside phosphorylase and xanthine oxidase are used as indicator enzymes. This method has been applied to the assay of nucleoside diphosphatase. Incidental to this work, the apparent Michaelis constant of phosphate for calf spleen purine nucleoside phosphorylase was determined to be 0.25 mm, and the extinction coefficient of uric acid at 293 nm and pH 7.4 was found to be 13.0 × 10³m^?1 cm^?1.     

Fiber type and temperature dependence of inorganic phosphate: implications for fatigue

Elevated levels of P_i are thought to cause a substantial proportion of the loss in muscular force and power output during fatigue from intense contractile activity. However, support for this hypothesis is based, in part, on data from skinned single fibers obtained at low temperatures (15°C). The effect of high (30 mM) P_i concentration on the contractile function of chemically skinned single fibers was examined at both low (15°C) and high (30°C) temperatures using fibers isolated from rat soleus (type I fibers) and gastrocnemius (type II fibers) muscles. Elevating P_i from 0 to 30 mM at saturating free Ca²⁺ levels depressed maximum isometric force (P_o) by 54% at 15°C and by 19% at 30°C (P < 0.05; significant interaction) in type I fibers. Similarly, the P_o of type II fibers was significantly more sensitive to high levels of P_i at the lower (50% decrease) vs. higher temperature (5% decrease). The maximal shortening velocity of both type I and type II fibers was not significantly affected by elevated P_i at either temperature. However, peak fiber power was depressed by 49% at 15°C but by only 16% at 30°C in type I fibers. Similarly, in type II fibers, peak power was depressed by 40 and 18% at 15 and 30°C, respectively. These data suggest that near physiological temperatures and at saturating levels of intracellular Ca²⁺, elevated levels of P_i contribute less to fatigue than might be inferred from data obtained at lower temperatures. skinned single fiber; force; power     

Improved estimation of potency in slope ratio assays

Regression coefficients are regarded as stochastic variables in order to account for this kind of variability. This is needed, because differences in responses at successive doses vary generally and thus the assumption of constancy of regression coefficients is violated.     

Fluorescent coupled enzyme assays for D-alanine: application to penicillin-binding protein and vancomycin activity assays

D-Alanine (D-Ala) is a ubiquitous constituent of bacterial cell walls. Assays for D-Ala can be used to investigate several aspects of cell wall biosynthesis and the effects of antibiotics on this process. High-sensitivity fluorescent assays for D-Ala were developed in a microtiter plate format based on d-aminoacid oxidase/horseradish peroxidase (DAO/HRP)-coupled reactions. For comparative purposes the classic chromogenic (UV-vis) assay using o-phenylenediamine (OPD) was also adapted to microtiter plates. OPD gave a lower limit of sensitivity of 2 nmol and was linear up to 60 nmol. Two commercially available fluorogenic HRP substrates were then tested in this assay. Amplex Red (AR) gave a lower limit of sensitivity of 2 pmol and was linear up to 400 pmol d-Ala. QuantaBlu (QB) based assays exhibited a lag in their response to D-Ala corresponding to 50 pmol D-Ala. This lag complicated calibration, but could be eliminated by addition of 150 pmol D-Ala to all assays. The QB assays were linear up to 3000 pmol D-Ala and gave a lower limit of sensitivity of 10 pmol. These assays are demonstrated for the characterization of the dd-carboxypeptidase activity of a soluble form of Escherichia coli penicillin-binding protein 5 (PBP 5) against the classic PBP substrate diacetyl-L-Lys-D-Ala-D-Ala. AR and QB based assays gave identical v/E(T) profiles, whereas OPD based assays gave slightly (10%) higher activity. This is consistent with the loss of a small amount of E. coli PBP 5 activity during the dilution necessary prior to its use in the highly sensitive fluorescent assays. These assays were then demonstrated for characterization of vancomycin binding to a D-Ala-D-Ala-based substrate.     

A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases

We present here an improvement in the classical molybdate method for inorganic phosphate determination. This method has high sensitivity, with 1 nmol of Pi giving an absorbance change of 0.060 A850 unit. It is highly reproducible and the color remains stable for at least 3.5 h. In addition the use of sodium dodecyl sulfate makes it possible to stop enzymatic reactions without organic phosphate hydrolysis. It also shows an extremely low interference by highly concentrated solutions of different origins. Of special interest is its high tolerance to protein, permitting as much as 50 mg/ml of human serum protein in the sample without precipitation or color interference. For these reasons, it proves to be very useful in the determination of ATPases in tissues such as the ocular lens with low specific activity.     

Polarimetry as a general method for enzyme assays.

Countercurrent distribution (CCD) and Martin-Synge distribution (MSD) were compared on the basis of the theory previously presented in this series. The comparison included the numbers of partition units required to obtain the same resolution degree of two compounds as well as the elution volumes and the widths of the elution curves. The ratio of the numbers of partition units, φ, was found to be proportional to αk₁ where α is the phase ratio and k₁ is the partition coefficient of the more rapidly moving component. The limit of φ when β → 1 was found equal to αk₁ + 1. Accordingly, in the case αk₁ ? 0, the methods possess approximately the same separation power, and in case αk₁ ? 1, about double the number of partition units is required in MSD as compared to CCD. In the case that αk_1,CCD = 1 and αk_1,MSD ? 0, the situation becomes roughly inversal. The ratio of the elution volumes, χ, in the two methods was found to be equal to φ in the case that the stationary phase volumes (ν_s) are equal and that the q = 1/(αk + 1) values are equal in the methods. On the same conditions, the ratio of the standard deviations of the elution curves, ψ, was found to be equal to φ^1/2/q^1/2, and the limit of ψ when β → 1 equal to 1/q₁. If, in addition, the condition αk₁ ? 0 is satisfied, ψ = 1. A practical comparison of the methods was also included, wherein attention was focused upon the real separation powers, the reproducibilities, the suitabilities for analytical or preparative purposes, the suitabilities for nonideal situations, the possibilities for automation, and the main structural features of some most important CCD and MSD apparatuses.     

Improved mean estimation and its application to diagonal discriminant analysis

MOTIVATION: High-dimensional data such as microarrays have created new challenges to traditional statistical methods. One such example is on class prediction with high-dimension, low-sample size data. Due to the small sample size, the sample mean estimates are usually unreliable. As a consequence, the performance of the class prediction methods using the sample mean may also be unsatisfactory. To obtain more accurate estimation of parameters some statistical methods, such as regularizations through shrinkage, are often desired. RESULTS: In this article, we investigate the family of shrinkage estimators for the mean value under the quadratic loss function. The optimal shrinkage parameter is proposed under the scenario when the sample size is fixed and the dimension is large. We then construct a shrinkage-based diagonal discriminant rule by replacing the sample mean by the proposed shrinkage mean. Finally, we demonstrate via simulation studies and real data analysis that the proposed shrinkage-based rule outperforms its original competitor in a wide range of settings.     

A method for preventing sorbitol interference with the determination of inorganic phosphate

A technique is described for preventing interference of sorbitol with the assay of P₁ by modifying the procedure of B. N. Ames (1966, in Methods in Enzymology, E. F. Neufeld and V. Ginsburg, eds., Vol. 8, pp. 115–118, Academic Press, New York). The new method relies on the ability of precipitated protein to bind phosphomolybdate and so allow separation of the P₁ from the soluble sorbitol. The conditions for the formation and precipitation of phosphomolybdate-protein complex and for the subsequent assay of P₁ are described. No unique set of conditions could be found which prevented interference at all sorbitol concentrations tested. Instead, conditions for the elimination of interference by particular sorbitol concentration ranges were established. The application of the procedure to samples containing 0–150 nmol of P₁ and 10–100 μmol of sorbitol is described. Complete recovery of P₁ was achieved after precipitation. Standard plots were linear. Coefficients of variation ranged from 9% with low amounts of P₁ (≤25 nmol) to 2.5% at higher levels (150 nmol). One hundred nanomoles of P₁ gave an absorbance at 700 nm of 0.87. Modifications are described to extend the technique to different sorbitol concentration ranges and other applications of the method are mentioned.