Dolbeare F A, Smith R E
Clin Chem. 1977 Aug;23(8):1485-91.
Enzyme activity can be measured in single cells or in cell suspensions by either static or flow microfluorometry when a fluorogenic substrate is used. We have used amino acid derivatives of arylamines as fluorogenic substrates for tagging cellular proteinases. The liberated aromatic amine, which can diffuse from the cell, is trapped as a fluorescent insoluble Schiff-base product with 5-nitrosalicylaldehyde, with the peak of fluorescence emission shifted from lambdaem 425 nm to lambdaem 530 and 595 nm. Although the reaction is faster at pH 4 than at higher pH's, the equilibrium during the assay of certain peptidase activities is such that the liberated aromatic amine is trapped in the cell at pH values as high as 7.5. 5-Nitrosalicylaldehyde causes almost no inhibition of substrate hydrolysis at 1 mmol/liter, a concentration exceeding that required for complete trapping of reaction product. The kinetics of enzymatic reactions with four synthetic substrates are demonstrated in intact Balb 3T3 cells and sonicated preparations in the presence of 5-nitrosalicylaldehyde. Hydrolytic rates for the substrate, CBZ-ala-arg-arg-4-methoxy-beta-naphthylamine, are given for single 3T3 cells by microfluorophotometry and for suspensions of 3T3 cells by flow cytometry. The clinical value of the method is demonstrated for differentiating mixed populations of leukocytes.
当使用荧光底物时，酶活性可以通过静态或流动微荧光法在单细胞或细胞悬液中进行测量。我们使用芳胺的氨基酸衍生物作为荧光底物来标记细胞蛋白酶。释放出的芳香胺可以从细胞中扩散出来，与5-硝基水杨醛形成荧光不溶性席夫碱产物而被捕获，荧光发射峰从λem 425 nm移至λem 530和595 nm。尽管在pH 4时反应比在更高pH值时更快，但在某些肽酶活性测定过程中的平衡使得释放出的芳香胺在pH高达7.5时仍被捕获在细胞中。5-硝基水杨醛在1 mmol/L的浓度下几乎不会抑制底物水解，该浓度超过了完全捕获反应产物所需的浓度。在完整的Balb 3T3细胞和超声破碎制剂中，在5-硝基水杨醛存在的情况下，展示了与四种合成底物的酶促反应动力学。通过微荧光光度法给出了单个3T3细胞对底物CBZ-ala-arg-arg-4-甲氧基-β-萘胺的水解速率，通过流式细胞术给出了3T3细胞悬液的水解速率。该方法在区分白细胞混合群体方面的临床价值得到了证明。
