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Title: Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-. beta. -naphthylamine derivatives

Abstract

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 lambda/sub em/ 425 nm to lambda/sub em/ 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 ofmore » leukocytes.« less

Authors:
;
Publication Date:
Research Org.:
Univ. of California, Livermore
OSTI Identifier:
7316519
Resource Type:
Journal Article
Journal Name:
Clin. Chem.; (United States)
Additional Journal Information:
Journal Volume: 23:8
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CELL FLOW SYSTEMS; PEPTIDE HYDROLASES; MEASURING METHODS; ALDEHYDES; AMINES; BIOCHEMICAL REACTION KINETICS; CYTOLOGICAL TECHNIQUES; FLUORESCENCE; LEUKOCYTES; PH VALUE; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; ENZYMES; HYDROLASES; KINETICS; LUMINESCENCE; ORGANIC COMPOUNDS; REACTION KINETICS; 550200* - Biochemistry

Citation Formats

Dolbeare, F A, and Smith, R E. Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-. beta. -naphthylamine derivatives. United States: N. p., 1977. Web.
Dolbeare, F A, & Smith, R E. Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-. beta. -naphthylamine derivatives. United States.
Dolbeare, F A, and Smith, R E. Mon . "Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-. beta. -naphthylamine derivatives". United States.
@article{osti_7316519,
title = {Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-. beta. -naphthylamine derivatives},
author = {Dolbeare, F A and Smith, R E},
abstractNote = {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 lambda/sub em/ 425 nm to lambda/sub em/ 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.},
doi = {},
url = {https://www.osti.gov/biblio/7316519}, journal = {Clin. Chem.; (United States)},
number = ,
volume = 23:8,
place = {United States},
year = {1977},
month = {8}
}