Van Duijn P, Van Noorden C J
Department of Cytochemistry and Cytometry, Sylvius Laboratories, University of Leiden, The Netherlands.
Histochem J. 1989 Sep-Oct;21(9-10):619-24. doi: 10.1007/BF01753363.
A simple and reproducible method was used for the cytophotometric assay of alkaline phosphatase activity by end point measurements after incubation at 70 degrees C. Alkaline phosphatase was incorporated in polyacrylamide gel model films and its activity was demonstrated with a simultaneous coupling method. The initial reaction rate was 4.7 times faster than at 37 degrees C. At 37 degrees C, linear reaction rates were obtained up to 90 min incubation. Deviation from linearity occurred only when the amount of final reaction product precipitated inside the films was too high to be measured cytophotometrically. In that case, levelling off of the reaction rate was due to the out-of-range error of the cytophotometer. At 70 degrees C, reaction rates were distinctly non-linear from the onset of incubation. This was due to heat inactivation of the enzyme molecules. A plateau level was reached after approximately 60 min incubation, irrespective of the amount of enzyme incorporated, indicating that all enzyme molecules had become inactivated after this incubation period. The inactivation process followed first-order kinetics. The plateau value as well as the slope of the initial reaction were found to be linearly related to the amount of enzyme incorporated. Therefore, plateau absorbance values can be used as a relative measure of enzyme activity instead of initial reaction rates. This type of measurement could be valuable for routine applications of enzyme cytochemistry in diagnostic pathology, or when cytochemical reaction products are used as markers in immunocytochemistry or hybridocytochemistry. Precise control of incubation time is not necessary once the plateau value has been reached and preparations can be mounted and measured later.
采用一种简单且可重复的方法，通过在70℃孵育后进行终点测量来进行碱性磷酸酶活性的细胞光度测定。碱性磷酸酶被掺入聚丙烯酰胺凝胶模型膜中，并通过同步偶联法显示其活性。初始反应速率比37℃时快4.7倍。在37℃下，孵育长达90分钟可获得线性反应速率。只有当膜内沉淀的最终反应产物量过高而无法通过细胞光度法测量时，才会出现偏离线性的情况。在这种情况下，反应速率趋于平稳是由于细胞光度计的超出范围误差。在70℃时，从孵育开始反应速率就明显呈非线性。这是由于酶分子的热失活。孵育约60分钟后达到平台水平，无论掺入的酶量如何，这表明在此孵育期后所有酶分子均已失活。失活过程遵循一级动力学。发现平台值以及初始反应的斜率与掺入的酶量呈线性相关。因此，平台吸光度值可用于作为酶活性的相对测量指标，而不是初始反应速率。这种测量类型对于酶细胞化学在诊断病理学中的常规应用，或者当细胞化学反应产物用作免疫细胞化学或杂交细胞化学中的标记物时可能具有价值。一旦达到平台值，就无需精确控制孵育时间，制备物可以稍后进行固定和测量。
