A continuous spectrophotometric assay for Clostridium histolyticum collagenase.

Abstract A continuously recording spectrophotometric assay has been developed for Clostridium histolyticum collagenase based on the hydrolysis of 2-furanacryloyl- l -leucylglycyl- l -prolyl- l -alanine (FALGPA). The hydrolysis of this peptide by collagenase obeys Michaelis-Menten kinetics with V = 1.8 × 105μkatal/kg and Km = 0.5 m m . FALGPA is hydrolyzed more rapidly by collagenase than any other commonly used synthetic substrate, but is not cleaved by any of the well-known proteinases such as trypsin, thermolysin, or elastase. The assay itself is rapid, convenient, and sensitive, and should greatly facilitate detailed kinetic studies of collagenase.

[1]  H. Noller,et al.  Spectrophotometric identification of acyl enzyme intermediates. , 1965, Biochemistry.

[2]  H. Noda,et al.  Kinetic studies on the action of collagenase. , 1965, Journal of biochemistry.

[3]  B. Johnson-Wint,et al.  A quantitative collagen film collagenase assay for large numbers of samples. , 1980, Analytical biochemistry.

[4]  J. Feder,et al.  Studies on the Bacillus subtilis neutral-protease- and Bacillus thermoproteolyticus thermolysin-catalyzed hydrolysis of dipeptide substrates. , 1970, Biochemistry.

[5]  J. Feder.  A spectrophotometric assay for neutral protease. , 1968, Biochemical and biophysical research communications.

[6]  G. Legler,et al.  [On proteins and their decomposition products. 18. The hydrolysis of synthetic and natural substrates by collagenase from Clostridium histolyticum]. , 1960, Hoppe-Seyler´s Zeitschrift für physiologische Chemie.

[7]  P. Davison,et al.  Tissue collagenase: a simplified, semiquantitative enzyme assay. , 1973, Analytical biochemistry.

[8]  J. Hunter,et al.  A neutral protease in rheumatoid synovial fluid capable of attacking the telopeptide regions of polymeric collagen fibrils. , 1975, Biochimica et biophysica acta.

[9]  I. Mandl,et al.  Clostridium histolyticum collagenase: its purification and properties. , 1958, Archives of biochemistry and biophysics.

[10]  S. Bernhard,et al.  Optical properties and the chemical nature of acyl-chymotrypsin linkages. , 1967, Journal of the American Chemical Society.

[11]  H. Noda,et al.  Hydrolysis of synthetic peptides by collagenase. , 1960, Biochimica et biophysica acta.

[12]  B. Vallee,et al.  Superactivation of thermolysin by acylation with amino acid N-hydroxysuccinimide esters. , 1975, Biochemistry.

[13]  T. Kono.  Purification and partial characterization of collagenolytic enzymes from Clostridium histolyticum. , 1968, Biochemistry.

[14]  R. Levenson.  A collagen film microassay for tissue collagenase. , 1976, Analytical biochemistry.

[15]  Y. Nagai,et al.  A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate. , 1976, Biochimica et biophysica acta.

[16]  A. Nordwig,et al.  [Quantitative colorimetric test for collagenase]. , 1960, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[17]  K. Nagasawa,et al.  Mode of action of bacterial collagenase on a synthetic substrate, (Pro-Pro-Gly)5. , 1979, Biochimica et biophysica acta.

[18]  G. Legler,et al.  [On proteins and their reduction products. XVI. On the specificity of collagenase from Clostridium histolyticum and its application to the determination of the primary structure of collagen. (Preliminary report)]. , 1959, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[19]  C. Franzblau,et al.  A new assay for collagenolytic activity. , 1978, Analytical biochemistry.

[20]  J. A. Yankeelov,et al.  Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors. , 1977, Biochimica et biophysica acta.

[21]  M. Nimni,et al.  A modified collagenase assay method based on the use of p-dioxane. , 1979, Analytical biochemistry.

[22]  G. W. Anderson,et al.  The Use of Esters of N-Hydroxysuccinimide in Peptide Synthesis , 1964 .

[23]  A. Kang,et al.  Studies on the specificity of bacterial collagenase. , 1970, Biochemical and biophysical research communications.

[24]  W. O. McClure,et al.  The reaction of carboxypeptidase A with chromophoric substrates. , 1966, Biochemistry.

[25]  J. Woessner,et al.  Mammalian collagenase: direct demonstration in homogenates of involuting rat uterus. , 1971, Biochemical and biophysical research communications.

[26]  P. Robertson,et al.  A reproducible quantitative collagenase radiofibril assay. , 1972, Clinica chimica acta; international journal of clinical chemistry.

[27]  W. Mitchell.  Pseudocollagenase: a protease from Clostridium histolyticum. , 1968, Biochimica et biophysica acta.

[28]  E. Wünsch,et al.  Zur quantitativen Bestimmung der Kollagenase , 1963 .

[29]  A. Barrett,et al.  Cathepsin B1. A lysosomal enzyme that degrades native collagen. , 1974, The Biochemical journal.

[30]  J. M. Marsh,et al.  A microassay for the direct demonstration of collagenolytic activity in Graafian follicles of the rat. , 1978, Biochimica et biophysica acta.

[31]  R. Rice,et al.  Radioactive labeling of proteins in vitro. , 1971, The Journal of biological chemistry.

[32]  H. Noda,et al.  The specificity of collagenase. , 1959, Biochimica et biophysica acta.

[33]  W. Grassman,et al.  [On the specificity of collagenase]. , 1959, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[34]  J. Uitto,et al.  Human leukocyte collagenase: characterization of enzyme kinetics by a new method. , 1977, Analytical biochemistry.

[35]  P. Hargrave,et al.  A sensitive assay for collagenolytic activity using tritiated collagen. , 1976, Analytical biochemistry.

[36]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[37]  B. McBride,et al.  A rapid sensitive collagenase assay. , 1975, Analytical biochemistry.

[38]  L. Sparrow,et al.  Isolation by affinity chromatography of neutral proteinase from Clostridium histolyticum. , 1973, Biochimica et biophysica acta.

[39]  P. Gallop,et al.  Studies on collagen. II. Properties of purified collagenase and its inhibition. , 1959, The Journal of biological chemistry.

[40]  J. Riordan,et al.  A continuous spectrophotometric assay for angiotensin converting enzyme. , 1979, Analytical biochemistry.

[41]  R. Feeney,et al.  The action of proteolytic enzymes on N,N-dimethyl proteins. Basis for a microassay for proteolytic enzymes. , 1969, The Journal of biological chemistry.

[42]  P. Bornstein.  Comparative sequence studies of rat skin and tendon collagen. I. Evidence for incomplete hydroxylation of individual prolyl residues in the normal proteins. , 1967, Biochemistry.