Chemical Stability of Indicators and Substrate-Indicators Used for the Detection of Phosphatase Activity

Abstract

Summary The reagents 2, 6-dibromo-N-chloro- p -quinoneimine (BQC), N, 2, 6-trichloro- p -quinoneimine (CQC), phenolphthalein phosphate, and p -nitrophenyl phosphate, in powder form and as solutions, for use as indicators or substrate-indicators in phosphatase testing, were stored under a variety of conditions, to observe rates of deterioration. CQC reagent was remarkably superior to BQC in maintaining chemical stability over long periods of time. Storage conditions for maximum stability of the four reagents were determined. BQC and CQC powder may be stored at any temperature between −5 and 20° C. for as long as 1 yr. BQC solutions may be stored at −5° C. for not longer than 30 days. CQC solutions may be stored at 5° C. for as long as 258 days. Phenolphthalein phosphate powder may be stored at −5° C. as long as 1 yr., whereas solutions of this reagent may be stored at −5° C. as long as 258 days. Powdered p -nitrophenyl phosphate may be stored at −5° C. as long as 105 days, and solutions of this reagent may be stored at −5° C. as long as 81 days. The indicators CQC and BQC, in solution, are best kept in dark brown, well-stoppered glass containers, while the two substrate-indicators are best protected in clear glass, well-stoppered containers. CQC and BQC were compared relative to their solubilities and rate and intensity of color development in simple model systems and in actual milk phosphatase systems. In absolute methanol, absolute ethanol, and 95% ethanol, CQC was rapidly and completely soluble. BQC was readily soluble only in absolute methanol. The rate of blue color development was influenced greatly by the chemical system in which the indicators reacted with available phenol. The rate was similar for both BQC and CQC in the zinc sulfate-borate color development system associated with the Sanders and Sager method (8). In the trichloracetic acid-carbonate buffer color development system of the Cornell phosphatase method, both indicators reacted more rapidly with the available phenol. Maximum color development for each indicator was attained four times faster at 37° C. and twice as fast at 25° C. in the trichloracetic acid-carbonate system. The data show that maximum color can be developed in the Cornell method in 15min. at room temperature, rather than the earlier recommended 15min. at 37° C. In the trichloroacetic acid-carbonate system CQC developed slightly more blue color than BQC at high levels of free phenol, but at low levels no differences were observed. The chemical stability, solubility, and rate and intensity of color development indicate that CQC is more suitable for use as an indicator for phosphatase testing than is BQC. It should receive preference in the future.