Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids

J Biol Chem. 1986 Aug 25;261(24):11282-9.


Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Body Fluids / analysis
  • Cathepsin B
  • Cathepsins / metabolism
  • Chromatography, Affinity
  • Cystatin B
  • Cystatin C
  • Cystatins*
  • Cysteine Proteinase Inhibitors
  • Electrophoresis, Polyacrylamide Gel
  • Half-Life
  • Humans
  • Kinetics
  • Kininogens / isolation & purification
  • Molecular Weight
  • Papain / metabolism
  • Protease Inhibitors / urine*
  • Proteins / isolation & purification*
  • Salivary Cystatins
  • Salivary Proteins and Peptides / isolation & purification


  • CST3 protein, human
  • CST4 protein, human
  • CSTB protein, human
  • Cystatin C
  • Cystatins
  • Cysteine Proteinase Inhibitors
  • Kininogens
  • Protease Inhibitors
  • Proteins
  • Salivary Cystatins
  • Salivary Proteins and Peptides
  • Cystatin B
  • Cathepsins
  • Cathepsin B
  • Papain