Structural organisations of hemoglobin and myoglobin influence their binding behaviour with phenothiazines

doi:10.1016/S0141-8130(98)00006-3

International Journal of Biological Macromolecules

Volume 23, Issue 1, July 1998, Pages 11-18

https://doi.org/10.1016/S0141-8130(98)00006-3 Get rights and content

Abstract

Binding modalities of chlorpromazine and trifluoperazine, two widely used antipsychotic phenothiazine drugs with hemoglobin and myoglobin have been studied to understand how the quaternary, tertiary and secondary structural organisations of the proteins regulate the binding process. NaCl-induced alteration in the quaternary structure of hemoglobin influences its binding modality with phenothiazines. Minor alterations in the tertiary structure of thermally denatured myoglobin (denaturation temperature ranging between 30–70°C) do not affect its affinity and the modality of binding with the drugs, but alterations in the secondary structure of the protein denatured at temperatures between 70–80°C influence its binding.

Introduction

Chlorpromazine (CPZ, molecular weight 315) and trifluoperazine (TFZ, molecular weight 407) are two non-planar phenothiazine drugs widely used in the treatment of psychoses [1]. TFZ, a derivative of phenothiazine has an extra hydrophobic group in its tail region and has three fluorine atoms instead of one chlorine atom in CPZ. TFZ is known to be potent from a therapeutic point of view at comparatively low doses and is inherently long acting [1]. The structures of the two drugs are shown in Fig. 1. One of the approaches in understanding the mechanism of drug action is to study its interaction with the functionally important macromolecular components of the cell on which the drug acts. CPZ is known to interact with DNA, RNA, proteins 2, 3and also with cell membranes [4].

Hemoglobin is the major protein component of red blood cell (RBC) and as an allosteric tetrameric protein, it has an important role in carrying oxygen from lungs to different tissues. Like hemoglobin, the muscle protein myoglobin is another heme-containing protein involved in the storage and transfer of oxygen within muscle cells [5]. α and β subunits of tetrameric hemoglobin (α₂β₂) and monomeric myoglobin are alike in the sense that each of them has eight α-helical segments, same percent (75%) of α-helix and similar three dimensional structural organisation. However, unlike quaternarily organised tetrameric hemoglobin with its four interacting subunits, monomeric myoglobin with its single polypeptide chain lacks subunit interaction property.

Quaternary organisation and the associated subunit interaction property of tetrameric hemoglobin confers it to have the regulatory property when small ligands like metabolites or drugs bind to it and modulate the functional activity of this important protein. In our previous report [6], we observed that at 0.15 M NaCl, hemoglobin predominantly existing as tetramer binds with CPZ exothermically and in positive cooperative mode, while monomeric myoglobin binds with the drug endothermically and in non-cooperative mode. This prompted us to investigate whether hemoglobin dissociated to its dimeric subunits and thus being partly devoid of quaternary organisation and loosing subunit interaction property, exhibits any change in modality of its binding with the drugs CPZ or TFZ, as compared to that in case of intact tetramer interacting with the respective drug, and hence to ascertain the importance of quaternary organisation of hemoglobin in its process of binding with the drug. The detailed thermodynamic and spectrofluorometric analyses of the binding features of CPZ or TFZ with hemoglobin at different NaCl molarity give clear evidences that tetrameric hemoglobin dissociated to its increasing dimeric forms has altered binding modality with each of the drugs.

The next obvious question that we have tried to address is how the tertiary and secondary organisation of the protein are involved in its process of binding with the drug. Studies on the interaction of CPZ or TFZ with heat-denatured myoglobin reveal that minor alterations in the tertiary structure of myoglobin due to denaturation at temperatures less than 70°C do not affect its binding behaviour with phenothiazines, but changes in the secondary structure of the protein denatured at temperature higher than 70°C significantly influence its binding modality with the drugs.

Section snippets

Phenothiazines

CPZ and TFZ were obtained as gifts from Sun Pharmaceuticals, India. Aqueous stock solutions of CPZ and TFZ were made fresh in a pyrex glass tube wrapped with black paper before each set of experiments. The concentrations of the drug solutions were determined spectrophotometrically from their respective molar extinction coefficients [7]using quartz cuvette: $ε^{CPZ}_{305nm} =4000 M^{−1} cm^{−1}, ε^{TFZ}_{308nm} =3162 M^{−1} cm^{−1}$

In the drug concentration range of our study (CPZ: 5–350 μM and TFZ: 5–100 μM), both the drug

Results and discussion

Fig. 2 is the plot of affinity constant (K) of CPZ or TFZ interaction with hemoglobin or myoglobin as a function of NaCl molarity ranging from 0.02 to 1.5 M. As the NaCl concentration is increased from 0.02 to 0.3 M, the binding affinity constants of both the drugs with hemoglobin gradually decrease, and thereafter from 0.3 to 1.5 M NaCl, the binding affinities gradually increase and tend to level off to a value close to that of CPZ or TFZ-myoglobin interactions. On the otherhand, the affinity

Acknowledgements

We thank the University Grants Commisson for providing a Senior Research Fellowship to Miss Jaya Bhattacharyya and the Council of Scientific and Industrial Research, for providing a Research Associateship to Dr Maitree Bhattacharyya. Thanks are due to Professor C.K. Dasgupta and the PD department of Bose Institute for making available to us the Hitachi F 3010 spectrofluorometer and Jasco 600 spectropolarimeter, respectively.

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¹: Present address: Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Calcutta 700 019, India

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Structural organisations of hemoglobin and myoglobin influence their binding behaviour with phenothiazines

Abstract

Introduction

Section snippets

Phenothiazines

Results and discussion

Acknowledgements

Chem Biol Interact

Biochimie

Biochem Pharmacol

Biochem Pharmacol

Int J Biol Macromol

Biochem Biophys Res Commun

J Biol Chem

Biochem Biophys Res Commun