Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

Title: Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

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Abstract

Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes. No Drawings

Inventors:: Smith, R E; Dolbeare, F A

Publication Date:: 1980-10-21

OSTI Identifier:: 7000909

Patent Number(s):: US 4229528; A

Application Number:: PPN: US 5-959555

Assignee:: PTO; EDB-94-125898

DOE Contract Number:: W-7405-ENG-48

Resource Type:: Patent

Resource Relation:: Patent File Date: 13 Nov 1978

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; PEPTIDE HYDROLASES; BIOASSAY; CHEMICAL ANALYSIS; FLUORESCENCE; SUBSTRATES; ENZYMES; HYDROLASES; LUMINESCENCE; ORGANIC COMPOUNDS; PROTEINS; 550200* - Biochemistry

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                    Smith, R E, and Dolbeare, F A. Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes.  United States: N. p., 1980. 
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                    Smith, R E, & Dolbeare, F A. Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes.  United States.

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                    Smith, R E, and Dolbeare, F A. Tue .  
        "Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes".  United States.

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@article{osti_7000909,

  title        = {Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes},

  author       = {Smith, R E and Dolbeare, F A},

  abstractNote = {Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes. No Drawings},

  doi          = {},

  journal      = {},
number       = ,

  volume       = ,

  place        = {United States},

  year         = {1980},

  month        = {10}

}

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Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

Patent Smith, Robert E. ; Dolbeare, Frank A.

Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.
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Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

Patent Smith, Robert E ; Dolbeare, Frank A

Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 5-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.
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Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-. beta. -naphthylamine derivatives

Journal Article Dolbeare, F A ; Smith, R E - Clin. Chem.; (United States)

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 ismore »« less
Fluorescent assay of proteinases in cultured mammalian cells

Journal Article Dolbeare, F ; Vanderlaan, M - J. Histochem. Cytochem.; (United States)

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.
DOI: 10.1177/27.11.512330
Unified standard substance for the photometric determination of aromatic amines and isocyanates

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In the determination of aromatic amines and isocyanates in solutions, air, washings from the skin, and extracts from uniforms, the compounds themselves are used as standard substances. The necessity of laborious purification of isocyanates and amines which are carcinogenic (benzidine, 2-naphthylamine, 2,4-toluidine) or are suspected of carcinogenicity (1-naphthylamine, toluidines, toluylenediamines, nitroanilines, and others) complicates the analysis and increases the sanitation hazard. The authors have suggested that noncarcinogenic (for humans) aniline (All-Union State Standard 5819-79) be used as the unified standard substance. The reaction of condensation with p-dimethylaminobenzaldehyde in acetic acid solution is used to determine the substances. The aniline fractionmore »« less

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