Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III /sup 3/H-acetylated collagens by bacterial and tissue collagenases

Journal Article · Sat Nov 01 00:00:00 EST 1986 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(86)90558-0· OSTI ID:5942411

Mallya, S K; Mookhtiar, K A; Van Wart, H E

Accurate and quantitative assays for the hydrolysis of soluble /sup 3/H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30/sup 0/C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters K/sub m/ and k/sub cat/ or V/sub max/. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.

• Research Organization: Florida State Univ., Tallahassee
• OSTI ID: 5942411
• Journal Information: Anal. Biochem.; (United States), Vol. 158:2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
COLLAGEN
ENZYMATIC HYDROLYSIS
BIOASSAY
ACETYLATION
CATTLE
CLOSTRIDIUM
MAN
PEPTIDE HYDROLASES
RATS
TRITIUM COMPOUNDS
ACYLATION
ANIMALS
BACTERIA
CHEMICAL REACTIONS
DECOMPOSITION
DOMESTIC ANIMALS
ENZYMES
HYDROLASES
HYDROLYSIS
LABELLED COMPOUNDS
LYSIS
MAMMALS
MICROORGANISMS
ORGANIC COMPOUNDS
PRIMATES
PROTEINS
RODENTS
RUMINANTS
SCLEROPROTEINS
SOLVOLYSIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques