Studies on the interaction of chlorpromazine with haemoglobin.

Abstract: 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 × 103 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 × 103M−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 (ΔH° = − 2.65 kcal/mol), whereas binding to myoglobin was endothermic (ΔH° = + 1.39 kcal/mol) with a high entropic contribution (ΔS° = + 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.

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.

Abstract: Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin : protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.

Abstract: Spectrophotometric and spectrofluorimetric studies reveal that an interaction occurs between hemoglobin and hematoporphyrin, a photosensitizing drug used in photodynamic therapy. Two concentration ranges of hematoporphyrin, 0.4–0.9 μM and 1.8–3.6 μM, representing significantly monomeric and aggregated (dimeric) state, respectively, have been used in the binding studies. The binding affinity constant ( K ) decreases, while the possible number of binding sites ( p ) increases as the concentration range of the porphyrin is increased. The nature of interaction has been studied by fluorescence quenching titration method under different ionic strengths and temperature conditions. It appears to be predominantly electrostatic and enthalpy-driven in the lower range of porphyrin concentration. However, the interaction follows mostly hydrophobic and entropy-driven modality in the higher concentration range of the ligand. The porphyrin-hemoglobin interaction results in release of oxygen from the protein. The extent of oxygen release depends on the stoichiometric ratio of hematoporphyrin: hemoglobin.

Abstract: Two important porphyrins, protoporphyrin IX and hematoporphyrin IX, derivatives of which form the basis of photosensitization in the photodynamic therapy of cancer treatment, interact with two physiologically important heme proteins hemoglobin and myoglobin. The extent and modality of these interactions vary with the state of aggregation of the two porphyrins. Upon binding with these proteins, both the drugs change the protein conformations and release the heme-bound oxygen from the oxyproteins. At the same time, the peroxidase activities of these proteins are potentiated due to the protein-porphyrin complexation, as is found in case of horseradish peroxidase also. The effect of porphyrins on heme proteins should be given due consideration in elucidating the details of the mechanism of porphyrin actions in therapy.

Abstract: 1. Human erythrocytes are protected or stabilized against hypotonic and mechanical hemolysis in the presence of low concentrations of many phenothiazines, reserpine, and haloperidol. At high concentrations all these surfactants cause lysis. 2. The erythrocyte stabilization by these compounds is long-lasting and depends on the concentration of erythrocytes. 3. The stabilizing potency correlates aproximately with the clinical potency of the phenothiazine. 4. Adsorption studies indicate that at maximal stabilization there is about 65 A 2 of erythrocyte membrane associated with one molecule of promethazine, 100 A 2 for chlorpromazine HCl, 140 A 2 for trifluoperazine diHCl, and 180 A 2 for fluphenazine diHCl. Since the area of the phenothiazine ring is about 50 A 2 , these values represent 90 to 25 per cent involvement of the membrane, if no adsorption to hemoglobin occurs. 5. The membrane stabilization is rapidly reversible. Lowering the extracellular drug concentration or photo-oxidizing the adsorbed drug causes the membrane to return to its original condition of fragility. 6. The prevention of hemolysis is also associated with the prevention of K + release; this distinguishes membrane stabilization from pro-lysis wherein K + is released. 7. Replacing isotonic sucrose by isotonic NaCl potentiates the lytic effect of phenothiazines. 8. The decrease in osmotic fragility (which corresponds to between one third and one half an atmosphere of pressure) may be explained possibly by an expansion of the cell membrane.

Abstract: Photosensitized damage to biological molecules is the initial process in phototoxic responses. It is now recognized that many phototoxic compounds can photosensitize damage to more than one type of biological substrate. The in vitro light-initiated reactions of phototoxic compounds with DNA, soluble proteins and membrane components can be classified by their molecular mechanisms: (1) those in which an excited state of the photo-toxic compound (or an unstable species derived from it) reacts directly with the biological substrate and (2) those in which a molecule derived from the phototoxic compound (a photoproduct or an activated oxygen species) reacts with the biological substrate. This paper describes the mechanisms by which chlorpromazine photosensitizes damage to membranes, protein and DNA and compares them to the mechanisms of photosensitization by psoralens, porphyrins, dyes, and other molecules.

Abstract: — The photoexcited chlorpromazine reacts with methanol to yield promazine and 2-methoxypromazine by two different reaction pathways: hydrogen atom abstraction and nucleophilic attack. respectively. When the photoexcitation of chlorpromazine is performed in the presence of protein or nucleic acids, chlorpromazine binds to the biopolymer. This binding is drastically pH-dependent and correlates to the phototoxic effect exhibited in chlorpromazine—photosensitization of E. coli. No photodynamic damage of E. coli attributed to CPZ-sensitization of molecular oxygen could be detected.

Abstract: In xeroderma pigmentosum, an inherited disorder of defective DNA repair, post-UV colony-forming ability of fibroblasts from patients in complementation groups A through F correlates with the patients' neurological status. The first xeroderma pigmentosum patient assigned to the recently discovered group G had the neurological abnormalities of XP. We have determined the post-UV colony-forming ability of cultured fibroblasts from this patient and from 5 more control donors. Log-phase fibroblasts were irradiated with 254 nm UV light from a germicidal lamp, trypsinized, and replated at known densities. After 2 to 4 weeks' incubation the cells were fixed, stained and scored for colony formation. The strains' post-UV colony-forming ability curves were obtained by plotting the log of the percent remaining post-UV colony-forming ability as a function of the UV dose. The post-UV colony-forming ability of 2 of the 5 new normal strains was in the previously defined control donor zone, but that of the other 3 extended down to the level of the most resistant xeroderma pigmentosum strain. The post-UV colony-forming ability curve of the group G fibroblasts was not significantly different from the curves of the group D fibroblast strains from patients with clinical histories similar to that of the group G patient.

Abstract: NMR spectra of the downfield region of normal adult hemoglobin are reported as a function of oxygenation and temperature Spectra were run in D2O at pD 74 A specially made NMR tube insert allowed precise measurement of the degree of oxygenation and of methemoglobin formation before and after taking the NMR spectrum Plots of the estimated intensity of the most downfield prominent NMR peak, identified as arising from a deoxy-beta subunit by Davis et al ((1971) J Mol Biol 60, 101-111), versus the average degree of oxygenation y, measured optically, yield a nearly straight line within experimental error, for samples stripped of organic phosphates and for samples containing 2,3-diphosphoglycerate or inositol hexaphosphate Intensities of peaks further upfield than this peak, previously attributed to deoxy-alpha subunits, are difficult to measure directly especially for samples containing inositol hexaphosphate The latter samples show broadening in these alpha peaks as the degree of oxygenation increases This extra broadening appears to increase with temperature Linearity of the beta peak intensity with oxygenation is expected if there is no large oxygen affinity difference between alpha and beta subunits However, the cooperativity of binding, and inaccuracy of the data, make it impossible to make accurate estimates of affinity differences