Fast-reacting thiols in rat hemoglobins can intercept damaging species in erythrocytes more efficiently than glutathione.
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.
Istituto di Clinica delle Malattie Nervose e Mentali, Sezione di Farmacologia, Università di Siena, 53100 Siena, and Centro di Biologia Molecolare, CNR, 00185 Roma, Italy.
R
R
Rossi
D
D
Barra
A
A
Bellelli
G
G
Boumis
S
S
Canofeni
P
P
Di Simplicio
L
L
Lusini
S
S
Pascarella
G
G
Amiconi
D000818:Animals; D003545:Cysteine / Q000378:metabolism; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D008297:Male; D008958:Models, Molecular; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004220:Disulfides; D006454:Hemoglobins; D016877:Oxidants; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D003958:Diamide; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D003545:Cysteine