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US4176009A
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Method of measuring collagenase activity
Assignee: Ajinomoto Co Inc
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Filing: 1977-11-21
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Granted: 1979-11-27
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Expires: 1997-11-21
Abstract
Abstract A method of measuring collagenase activity using as substrates peptide derivatives having the structure A-Pro-B-Gly-C-Ala-Gly-E wherein A is a hydrophobic neutral or acidic chromophore, B is an amino acid residue, C is Ile or Leu, E is Gln-D-Arg-OH or D-Arg-OH and all the amino acid residues except for Gly are L-configuration unless otherwise stated.
Inventors
- 👤 Shumpei Sakakibara
- 👤 Yutaka Nagai
- 👤 Kenji Fujiwara
- 👤 Takahiro Sakai
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 10:08
CPC Classifications
C; C07; C07K; C07K5/00; C07K5/04; C07K5/08; C07K5/0802; C07K5/0804; C07K5/0808; C07K14/00; C07K14/435; C07K14/78; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/28
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
120 words
A method of measuring collagenase activity which comprises contacting a compound having the structure A-Pro-B-Gly-C-Ala-Gly-E with collagenase in an aqueous medium whereby said compound is hydrolyzed to release peptide fragments, extracting the released peptide fragments containing A with an organic solvent, and assaying the extract by subjecting the extract to colorimetry, said A being a hydrophobic, and neutral or acidic chromophore selected from the group consisting of 2,4-dinitrophenyl, 5-dimethyl-amino-1-naphthalene-sulfonyl, p-phenylazobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-(4-hydroxy-1-naphthylazo)-benzenesulfonyl and p-phenylazobenzoyl, said B being an amino acid residue of an alpha-amino acid having up to 15 carbon atoms occurring in protein, said C being Ile or Leu, said E being Gln-D-Arg-OH or D-Arg-OH, and all the amino acid residues except for Gly being L-configuration unless otherwise stated.
Claim 2
Independent Claim
120 words
A method of measuring collagenase activity which comprises contacting a compound having the structure A-Pro-B-Gly-C-Ala-Gly-E with collagenase in an aqueous medium whereby said compound is hydrolyzed to release peptide fragments, extracting the released peptide fragments containing A with an organic solvent, and assaying the extract by subjecting the extract to colorimetry, said A being a hydrophobic, and neutral or acidic chromophore selected from the group consisting of 2,4-dinitrophenyl, 5-dimethyl-amino-1-naphthalene-sulfonyl, p-phenylazobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-(4-hydroxy-1-naphthylazo)-benzenesulfonyl and p-phenylazobenzoyl, said B being an amino acid residue of an alpha-amino acid having up to 15 carbon atoms occurring in protein, said C being Ile or Leu, said E being Gln-D-Arg-OH or D-Arg-OH, and all the amino acid residues except for Gly being L-configuration unless otherwise stated.
Claim 3
Dependent Claim
30 words
A method as set forth in claim 1, wherein said contacting is carried out at a temperature of 30° to 45° C. and at a pH of 6 to 9.
Claim 4
Dependent Claim
40 words
A method as set forth in claim 2, wherein said A is 2,4-dinitrophenyl, said B is a residue of a compound selected from the group consisting of alanine, leucine and glutamine, said C is Ile and said E is Gln-D-Arg-OH.
Claim 5
Dependent Claim
32 words
A method as set forth in claim 1 wherein said collagenase activity is calculated by subtracting from the absorbance obtained by said colorimetry the absorbance obtained by substituting said compound by A-B-Gly-C-Ala-Gly-E.
Claim 6
Dependent Claim
27 words
A method as set forth in claim 4, wherein said A is 2,4-dinitrophenyl, said B is Gln or Ala, said C is Ile and E is Gln-D-Arg-OH.
Claim 7
Dependent Claim
32 words
A method as set forth in claim 2, wherein said collagenase activity is calculated by subtracting from the absorbance obtained by said colorimetry the absorbance obtained by substituting said compound by A-B-Gly-C-Ala-Gly-E.
Claim 8
Dependent Claim
23 words
A method as set forth in claim 2, wherein A is a compound selected from the group consisting of 2,4-dinitrophenyl, p-(4-hydroxy-1-napthylazo)-benzenesulfonyl and p-phenylazobenzoyl.
Claim 9
Dependent Claim
41 words
A method as set forth in claim 1, wherein B is a residue of a compound selected from the group consisting of glycine, alanine, valine, leucine, serine, threonine, proline, phenylalanine, tyrosine, tryptophane, lysine, arginine, aspartic acid, asparagine, glutamic acid and glutamine.
Claim 10
Dependent Claim
27 words
A method as set forth in claim 1, wherein B is a residue of a compound selected from the group consisting of alanine, proline, leucine and glutamine.
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) | 1982-11-26 | 1983-05-28 | 1983-11-27 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1986-11-26 | 1987-05-28 | 1987-11-27 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1990-11-26 | 1991-05-28 | 1991-11-27 | $7700.00 | Window Closed / Lapsed |