Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes
OwnerRobert E. Smith, Frank A. Dolbeareinventors
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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.
Claims5
A method of assaying for enzymes that hydrolyse an arylamide bond comprising:
- (a) adding to an unknown to be tested for an enzyme that hydrolyzes an arylamide bond an amino acid-containing, water dispersible, aromatic amine substrate which is selected so that when hydrolyzed by the enzyme, it produces an aromatic amine from the group consisting of 4-methoxy-2-naphthylamine, 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and 7-amino-4-methylcoumarin;
- (b) incubating the mixture for a time sufficient to allow reaction of the enzyme with the substrate;
- (c) adding to the unknown, either before or after incubating, a water dispersible aromatic aldehyde, selected from the group consisting of benzaldehyde, p-nitrobenzaldehyde and 5-nitrosalicylaldehyde, suitable for coupling to the hydrolyzed aromatic amine to produce a fluorescent complex with the aromatic amine;
- (d) exposing the aldehyde-containing, incubated mixture to ultraviolet light;
- and
- (e) sensing the fluorescence of said exposed mixture for a time sufficient.
Dependencies are inferred from the claim text.