US4229528A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes
Assignee: Individual
•
Filing: 1978-11-13
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Granted: 1980-10-21
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Expires: 1998-11-13
Abstract
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.
Inventors
- 👤 Robert E. Smith
- 👤 Frank A. Dolbeare
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 15:31
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37
Claims Analysis
Total Claims: 6
Claim 1
Independent Claim
147 words
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.
Claim 2
Independent Claim
147 words
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.
Claim 3
Dependent Claim
15 words
The method of claim 1 in which said sensing is accomplished through an optical filter.
Claim 4
Dependent Claim
17 words
The method of claim 2 in which said sensing is in the orange-red region of the spectrum.
Claim 5
Dependent Claim
12 words
The method of claim 1 in which said adding is before incubating.
Claim 6
Dependent Claim
22 words
The method of claim 1 wherein said aromatic aldehyde selected is 5-nitrosalicylaldehyde and said aromatic amine substrate selected produces 4-methoxy-2-naphthylamine when hydrolysed.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1983-10-21 | 1984-04-21 | 1984-10-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1987-10-21 | 1988-04-21 | 1988-10-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1991-10-21 | 1992-04-21 | 1992-10-21 | $7700.00 | Window Closed / Lapsed |