Removal of Arg141 from the alpha chain of human hemoglobin by carboxypeptidases N and M

Circ Res. 1996 Apr;78(4):635-42. doi: 10.1161/01.res.78.4.635.


Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (alpha-Arg141) of the alpha chain of human adult hemoglobin. An arginase contamination present in the hemoglobin preparation, which converted the released arginine to ornithine, was removed by gel filtration. CPM was about 20 times more efficient than CPN or its active subunit in hydrolyzing oxyhemoglobin and cleaved oxyhemoglobin twice as fast as deoxyhemoglobin. The hydrolysis of the peptide bond of alpha-Arg141 accelerated the dissociation rate of the tetramer deoxy-des-alpha-Arg141 hemoglobin to dimers 2500-fold over that of deoxyhemoglobin, as measured by haptoglobin binding. Moreover, the dissociation of the deoxy-des-alpha-Arg141 hemoglobin tetramer to dimers was not affected by 2,3-diphosphoglyceric acid. Des-alpha-Arg141 hemoglobin had a higher oxygen affinity (P50, 5.51 mm Hg; control, 19.94 mm Hg [P50 is the partial pressure of oxygen that gives 50% of the saturation of hemoglobin]) and a lower apparent cooperativity (Hill coefficient: n, 1.02; control, 2.24) than unhydrolyzed hemoglobin. After hemoglobin was incubated in human plasma, its oxygen-binding parameters, the P50, and the Hill coefficient decreased drastically due to cleavage by CPN. In the perfused rat heart, des-alpha-Arg141 hemoglobin was a more effective coronary vasoconstrictor than hemoglobin, possibly because it dissociated to dimers in the coronary vascular bed. A covalently cross-linked hemoglobin was less active than native hemoglobin. The coronary vasoconstriction was caused by multiple factors, including interference with vasodilation by nitric oxide and eicosanoids. Thus, the hydrolysis of hemoglobin by CPM and CPN demonstrated the contribution of the alpha-Arg141 residue to sustaining the tetrameric structure of hemoglobin and its normal oxygen affinity and vasoactivity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adult
  • Animals
  • Arteries / drug effects
  • Coronary Vessels / drug effects
  • Cross-Linking Reagents / pharmacology
  • GPI-Linked Proteins
  • Hemoglobins / drug effects
  • Hemoglobins / metabolism*
  • Humans
  • Hydrolysis
  • Kinetics
  • Lysine Carboxypeptidase / metabolism*
  • Male
  • Metalloendopeptidases / metabolism*
  • Oxygen / metabolism
  • Oxyhemoglobins / metabolism
  • Rats
  • Rats, Sprague-Dawley


  • Cross-Linking Reagents
  • GPI-Linked Proteins
  • Hemoglobins
  • Oxyhemoglobins
  • deoxy-des-alpha-arginine(141) hemoglobin
  • deoxyhemoglobin
  • carboxypeptidase M
  • Lysine Carboxypeptidase
  • Metalloendopeptidases
  • Oxygen