An evaluation of fluorometric proteinase assays which employ fluorescamine

Christopher H Evans, John D. Ridella

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37°C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.

Original languageEnglish (US)
Pages (from-to)411-420
Number of pages10
JournalAnalytical Biochemistry
Volume142
Issue number2
DOIs
StatePublished - Nov 1 1984
Externally publishedYes

Fingerprint

Microbial Collagenase
Fluorescamine
Assays
Peptide Hydrolases
Gelatinases
Collagenases
Gelatin
Conditioned Culture Medium
Caseins
Trypsin
Amines
Collagen
Substrates
Enzymes
Tissue

Keywords

  • collagenase
  • fluorescence
  • proteases
  • synovial cell culture
  • trypsin

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

An evaluation of fluorometric proteinase assays which employ fluorescamine. / Evans, Christopher H; Ridella, John D.

In: Analytical Biochemistry, Vol. 142, No. 2, 01.11.1984, p. 411-420.

Research output: Contribution to journalArticle

@article{ab52e67ddc3b4e1a83c42eb5ab7969be,
title = "An evaluation of fluorometric proteinase assays which employ fluorescamine",
abstract = "The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37°C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.",
keywords = "collagenase, fluorescence, proteases, synovial cell culture, trypsin",
author = "Evans, {Christopher H} and Ridella, {John D.}",
year = "1984",
month = "11",
day = "1",
doi = "10.1016/0003-2697(84)90485-8",
language = "English (US)",
volume = "142",
pages = "411--420",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - An evaluation of fluorometric proteinase assays which employ fluorescamine

AU - Evans, Christopher H

AU - Ridella, John D.

PY - 1984/11/1

Y1 - 1984/11/1

N2 - The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37°C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.

AB - The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37°C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.

KW - collagenase

KW - fluorescence

KW - proteases

KW - synovial cell culture

KW - trypsin

UR - http://www.scopus.com/inward/record.url?scp=0021737339&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021737339&partnerID=8YFLogxK

U2 - 10.1016/0003-2697(84)90485-8

DO - 10.1016/0003-2697(84)90485-8

M3 - Article

C2 - 6099062

AN - SCOPUS:0021737339

VL - 142

SP - 411

EP - 420

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -