Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method

Eur J Biochem. 1985 May 2;148(3):447-53. doi: 10.1111/j.1432-1033.1985.tb08860.x.


A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with Km values of 4.0 microM for folate, 8.0 microM for methotrexate and 34.0 microM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Carboxypeptidases / isolation & purification*
  • Chemical Phenomena
  • Chemistry
  • Chromatography, DEAE-Cellulose
  • Chromatography, Ion Exchange
  • Coloring Agents*
  • Enzyme Activation / drug effects
  • Hydrogen-Ion Concentration
  • Kinetics
  • Metals / pharmacology
  • Molecular Conformation
  • Pseudomonas / enzymology*
  • Pseudomonas / growth & development
  • Triazines*
  • Zinc / pharmacology
  • gamma-Glutamyl Hydrolase / isolation & purification*


  • Coloring Agents
  • Metals
  • Triazines
  • Carboxypeptidases
  • gamma-Glutamyl Hydrolase
  • Zinc