Fast-reacting thiols in rat hemoglobins can intercept damaging species in erythrocytes more efficiently than glutathione

J Biol Chem. 1998 Jul 24;273(30):19198-206. doi: 10.1074/jbc.273.30.19198.


The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mM), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t1/2 <100 ms; slow reacting (sHbSH), with t1/2 30-50 s; and very slow reacting (vsHbSH), with t1/2 180-270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125beta(H3)) conjugates with DTNB 100 times faster than glutathione and approximately 4000 times more rapidly than (v)sHbSH (Cys-13alpha(A11) and Cys-93beta(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (approximately 6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123beta(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mM, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cysteine / metabolism
  • Diamide / pharmacology
  • Disulfides / metabolism
  • Dithionitrobenzoic Acid / pharmacology
  • Erythrocytes / drug effects
  • Erythrocytes / metabolism*
  • Glutathione / metabolism*
  • Hemoglobins / metabolism*
  • Male
  • Models, Molecular
  • Oxidants / pharmacology
  • Oxidative Stress
  • Protein Conformation
  • Rats
  • Rats, Sprague-Dawley
  • Structure-Activity Relationship
  • Sulfhydryl Compounds / metabolism*
  • Sulfhydryl Reagents / pharmacology


  • Disulfides
  • Hemoglobins
  • Oxidants
  • Sulfhydryl Compounds
  • Sulfhydryl Reagents
  • Diamide
  • Dithionitrobenzoic Acid
  • Glutathione
  • Cysteine