Ramendra K. Poddar

Evidence for cooperative binding of chlorpromazine with hemoglobin: Equilibrium dialysis, fluorescence quenching and oxygen release study

Binding of chlorpromazine (CPZ) with human hemoglobin has been studied by equilibrium dialysis and fluorescence quenching. Results of equilibrium dialysis experiment when analysed by Hill plot revealed that the binding was positively cooperative with overall affinity constant K = 3.8 x 10(3) M-1. CPZ quenched the fluorescence of hemoglobin and the analysis of the quenching data by Stern-Volmer equation indicated two types of quenching process, namely, dynamic and static quenching. Dynamic quenching constant was measured from the decay of fluorescent life time of tryptophans of hemoglobin in presence of CPZ. Static quenching constant concerned with the ground state complex formation between CPZ and hemoglobin was found to be 5 x 10(3) M-1. Almost all the tryptophans of hemoglobin were found to be accessible for CPZ to interact with. Oxygen was found to be released when CPZ was added to hemoglobin. Extent of release of oxygen depends on the D/P ratio of CPZ(D): hemoglobin(P).

Interaction of chlorpromazine with myoglobin and hemoglobin: A comparative study

The mode and nature of the binding of chlorpromazine (CPZ), a psychotropic drug, with myoglobin, a monomeric muscle protein, were studied spectrofluorometrically and the results compared with those from the binding of CPZ to hemoglobin, a tetrameric allosteric protein from red blood cells (RBC). CPZ interacted with myoglobin in a non-cooperative mode, with a binding constant of 8.4 x 10(3) M-1 in 0.145 M NaCl, pH 6.8, whereas in the case of hemoglobin this interaction was found to be positively cooperative with a binding constant of 4.2 x 10(3) M-1. The interaction of CPZ with myoglobin was not influenced by the NaCl molarity of the solution, whereas CPZ interaction with hemoglobin significantly decreased with increasing NaCl molarity, indicating that CPZ-hemoglobin binding is mostly electrostatic in nature, whereas that of the CPZ-myoglobin complex is of a non-electrostatic type. Thermodynamic analysis revealed that binding of CPZ to hemoglobin was exothermic (delta H degrees = -2.65 kcal/mol), whereas binding to myoglobin was endothermic (delta H degrees = + 1.39 kcal/mol) with a high entropic contribution (delta S degrees = +23 cal/degree/mol), suggesting that CPZ binding to myoglobin is hydrophobic in nature. Such contrasting binding features of this drug have been discussed in the light of a typical subunit interaction property present and absent in hemoglobin and myoglobin, respectively.

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

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 degrees 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 degrees C influence its binding.

Induction of apoptosis by Phenothiazine derivatives in V79 cells.

Phenothiazine derivatives chlorpromazine (cpz) and trifluoperazine (tfp) were found to induce apoptosis, abnormal cell cycle and expression of p53 in Chinese hamster lung fibroblast V79 cells. Both the drugs can induce apoptosis when cells are treated with drug at a concentration of 10 microg/ml within 4 h, as detected by propidium iodide staining and DNA fragmentation analysis. Flow cytometric analysis revealed that the apoptotic response is mediated by a loss of G(1) population of cells. In Western blot analysis, p21 is induced and p53 is accompanied by additional bands. Also indirect immunolabeling of single cells revealed that p21 is accumulated from cytoplasm into nucleus after the drug treatment and the intensities of p53 increased. Our findings demonstrate for the first time that phenothiazine derivatives, in addition to their cytotoxic effects, could induce apoptosis, an observation that has important clinical implications.

Cytotoxic and genetic effects of X-irradiation of human cells in the presence of chlorpromazine

Human epidermoid carcinoma cells (Hep-2) were X-irradiated in the presence of 5-10 micrograms/ml of chlorpromazine (CPZ). Survival of the cells decreased with increasing CPZ concentration. Lymphocytes from three normal volunteers exposed to X-irradiation in the presence of CPZ showed an increased frequency of dicentric and ring formation.

Studies on the interaction of chlorpromazine with haemoglobin

The interaction of chlorpromazine (CPZ), a widely used antipsychotic tranquillizer, with the allosteric protein haemoglobin, has been studied by different methods. From r versus Cf plot obtained by an equilibrium dialysis experiment, the maximum value of r was found to be 6.8 at 0.15 M NaCl. Binding parameters, namely the affinity constant K and the degree of cooperativity nH, were determined from the Hill plot. Circular dichroism studies indicate a conformation change of haemoglobin in the presence of CPZ. Oxygen has been found to be released from haemoglobin with the progressive addition of CPZ. The extent of the release of oxygen depends on the stoichiometric ratio of CPZ: haemoglobin (D/P). The possible nature of the binding site of the protein has been discussed on the basis of the information obtained from fluorescence measurements.

Trifluoperazine is More Effective than Chlorpromazine in Releasing Oxygen from Haemoglobin and Myoglobin

The extent of oxygen release from two heme proteins, haemoglobin and myoglobin have been studied in the presence of trifluoperazine and chlorpromazine (5–1000 μM).

Chlorpromazine induced aggregation of normal human hemoglobin. Electron microscopic evidence

Normal human hemoglobin exceeding a certain minimum concentration (called critical aggregation concentration) undergoes aggregation in presence of the psychotherapeutic drug chlorpromazine (CPZ). The critical aggregation concentration decreases with the increase of CPZ concentration. Electron micrographs of CPZ-treated hemoglobin clearly indicate that the aggregates of hemoglobin are in filamentous form of average width 75 +/- 8 A. A possible mechanism for such aggregation has been discussed.