Tertiary conformational transition constant of guinea pig haemoglobin determined from the reaction of 5,5′-dithiobis (2-nitrobenzoate) with CysF9[93]β and CysH3[125]β

Idowu Adeogun, Olaniyi Yusuff, Kehinde Okonjo


We have determined Kequ, the equilibrium constant for the reaction of 5,5′-dithiobis(2-nitrobenzoate) — DTNB — with the CysF9*93+β and CysH3*125+β sulphydryl groups of various derivatives of guinea pig haemoglobin at 25oC. In the pH range 5.6 to 9, Kequ decreases almost 50-fold: from a mean of 3.45 ± 0.2 to a mean of 0.073 ± 0.01. Quantitative analyses of the pH dependence profiles of Kequ enable the determination of Krt, the equilibrium constant for the rt tertiary conformational transition of haemoglobin. The t isomer population is 53.9 (± 2)%. In the r conformation the pKas of the amino acid residues whose ionisations are coupled to the reaction of DTNB with the sulphydryl groups are 5.74 ± 0.02 — for a combination of HisNA2*2+β and HisH21*143+β) — and 7.74 ± 0.2 for ValNA1*1+β); in the t conformation they are 5.88 ± 0.05 and 8.23 ± 0.1, respectively.
Keywords --- guinea pig haemoglobin; sulphydryl groups; 5,5′-dithiobis(2-nitrobenzoate); equilibrium constants; tertiary conformational transition.

Full Text:



Miranda, J. J., Highly reactive cysteine residues in rodent hemoglobins, Biochem. Biophys. Res. Commun. 275 (2000) 517–523.

Kleinman, W. A. Komninou, D., Leutzinger, Y., Colosimo, S., Cox, J., Lang, C. A., Richie, J. P., Protein glutathiolation in human blood, Biochem. Pharmacol. 65 (2003) 741–746.

. Rossi, R., Barra, D., Bellelli, A., Boumis, G., Canofeni, S., Di Simplicio, P., Lusini, L., Pascarella, S., Amiconi, G. Fast-reacting thiols in rat hemoglobins can intercept damaging species in erythrocytes more efficiently than glutathione, J. Biol. Chem. 273 (1998) 19198–19206.

Chan, N. L., Rogers, P. H., Arnone, A. Crystal structure of the S-nitroso form of liganded human hemoglobin, Biochemistry 37 (1998) 16459–16464.

Guidotti, G. The rates of the reaction of the sulphydryl groups of human haemoglobin, J. Biol. Chem. 240 (1965) 3924-3927.

Antonini, E., Brunori, M. On the rate of conformation change associated with ligand binding in haemoglobin, J. Biol. Chem. 244 (1969) 3909– 3912.

Gibson, Q. H. p-Mercuribenzoate as an indicator of conformation change in hemoglobin, J. Biol. Chem. 248 (1973) 1281–1284.

Perutz, M.F., Fersht, A.R., Simon, S.R., Roberts, G.C.K. Influence of globin structure on the state of the heme. II. Allosteric transitions in methemoglobin, Biochemistry 13 (1974) 2174– 2286.

Hensley, P., Edelstein, S.J., Wharton, D.C., Gibson, Q.H. Conformation and spin state in methemoglobin, J. Biol. Chem. 250 (1975) 952– 960.

Heidner, E. J., Lardner, R.C., Perutz, M.F. Structure of horse carbonmonoxy- haemoglobin, J. Mol. Biol. 104 (1976) 707–722.

Baldwin, J. M. The structure of human carbonmonoxyhaemoglobin at 2.7Å resolution, J. Mol. Biol. 136 (1980) 103–128.

Shaanan, B. Structure of human oxyhaemoglobin at 2.1 Å resolution, J. Mol. Biol. 171(1983) 31– 59.

Okonjo, K.O., Vega-Catalan, F.J., Ubochi, C.I. Temperature-jump studies on haemoglobin: Kinetic evidence for a non-quaternary isomerization process in deoxy- and carbonmonoxyhaemoglobin, J. Mol. Biol. 208 (1989) 347– 354.

Okonjo, K.O., Fodeke, A.A. Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: acetylation of NH3+ terminal group of the β chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form, Biophys. Chem. 119 (2006) 196–204.

Okonjo, K.O., Fodeke, A.A., Kehinde, A.T. Reversible reaction of 5,5′ dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: Variation of the equilibrium and reverse rate constant with pH, Biophys. Chem. 121 (2006) 65 – 73.

Okonjo, K. O., Adediji, T. A., Fodeke, A. A., Adeboye, O., Ezeh, C. V. Transition of hemoglobin between two tertiary conformations: determination of equilibrium and thermodynamic parameters from the reaction of 5,5′ dithiobis(2-nitrobenzoate) with the CysF9*93+β sulfhydryl group, Biophys. Chem. 128 (2007) 56–62.

Okonjo, K.O., Bello, O.S., Babalola, J.O. Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica), Biochim. Biophys. Acta 1784 (2008) 464–471.

Okonjo, K. O., Adeogun, I., Babalola, J. O. Transition of haemoglobin between two tertiary conformations: Inositol hexakisphosphate increases the transition constant and the affinity of sheep haemoglobin for 5,5′-dithiobis(2-nitrobenzoate), Biochim. Biophys. Acta 1794 (2009) 398–409.

Okonjo, K.O., Adeogun, I., Babalola, J.O. Tertiary conformational transition in sheep haemoglobins induced by reaction with 5,5´-dithiobis(2-nitrobenzoate) and by binding of inositol hexakisphosphate, Biophys. Chem. 146 (2010) 65- 75.

Okonjo, K.O., Aboluwoye, C.O., Babalola, J.O., Usanga, I.A. Organic phosphate binding groups electrostatically linked to the reactivity of the CysF9[93]β sulphydryl group of haemoglobin, J. Chem. Soc., Faraday Trans., 91 (1995) 2095–2100.

Okonjo, K. O., Aken’ova, Y. A., Aboluwoye, C. O., Nwozo, S. , Akhigbe, F. U., Babalola, J. O., Babarinde, N. A. Effect of A3[6]βGlu→Val mutation on the reactivity of CysF9[93]β sulphydryl group of human haemoglobin S, J. Chem. Soc., Faraday Trans., 92 (1996) 1739–1746.

Okonjo, K.O., Okia, T.O. Hemoglobins with multiple reactive sulphydryl groups: The reaction of pigeon hemoglobin with 5,5'-dithiobis(2-nitrobenzoic acid), J. Protein Chem. 12 . (1993) 639-646.

Okonjo, K.O., Adejoro, I.A. Hemoglobins with multiple reactive sulphydryl groups: The reaction of dog hemoglobin with 5,5'-dithiobis(2-nitrobenzoic acid), J. Protein Chem. 12 (1993) 33-37.

Babalola, J.O., Uko, L.U., Okonjo, K.O., an Eldik, R. Hemoglobins with multiple reactive sulphydryl groups: Reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with guinea pig hemoglobin, Bulletin of the Chemical Society of Ethiopia 16 (2002) 175-186.

Yusuff, K.O. Equilibrium studies of the reaction of 5,5′-dithiobis(2-nitrobenzoate) with guinea pig haemoglobin, MSc thesis, University of Ibadan (2005).

Arnone, A. X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemoglobin, Nature 237 (1972) 146-148.

Arnone, A., Perutz, M.F Structure of inositol hexaphosphate-human deoxyhaemoglobin complex, Nature 249 (1974) 34-36


  • There are currently no refbacks.