Interaction of antimalarial drugs with hemin

Hemin (ferriprotoporphyrin IX) is shown to form complexes with the chloroquine class of antimalarial drugs. The Soret band of hemin becomes optically active upon the addition of chiral drugs. Results on the hemin-induced quenching of the fluorescence of chloroquine are consistent with the formation of a 2:1 hemin:drug complex with a formation constant of 1.4 x 10(7) at 298 K. Also a direct comparison of the drug-treated and drug-free parasites themselves, by the noninvasive photoacoustic spectroscopic method, reveals an in vivo interaction between endogenous hemin and the added drug.     

Interaction of hemin with quadruplex DNA

A DNA enzyme with peroxidase activity is a G-quadruplex-based DNAzyme formed by hemin and G-quadruplex DNA. Activity of peroxide DNAzymes can be influenced by the structure of quadruplex DNA. In this investigation, the interaction of hemin with T30695 G-quadruplex DNA is evaluated. Molecular dynamic simulation indicates that the binding mode of hemin to G-quadruplex DNA is end-stacking, which is consistent with absorption spectroscopy. Based on fluorescence spectroscopy, hemin ejects thiazole orange from bases of four-strand DNA. Circular dichroism spectra showed that no alteration occurs in this type of DNA structure.

Open image in new window

Graphical Abstract Peroxidase DNAzyme is formed by hemin and G-quadruplex DNA.

     

Interaction of spectrin with hydrophobic agaroses

Purified spectrin was found to interact strongly with hydrophobic agaroses such as Phenyl- or Octyl-Sepharose, in the presence of EDTA. From the complexes formed spectrin was eluted with ethylene glycol but not with low ionic strength solutions. The binding capacity of spectrin increased with increasing ionic strength of the equilibration buffer and showed but little dependence on its pH value. The fragments obtained by proteolysis of spectrin carried out under mild conditions were also found to bind strongly to phenyl-agarose, and were eluted with ethylene glycol. The fractions eluted with ethylene glycol contained two closely related polypeptides of Mr 65,000 and 60,000.     

Association of hemin with protein 4.1 as compared to spectrin and actin

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 . 10(7) M-1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.     

Interaction of hemin with placental glutathione transferase

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.     

Interaction of spectrin with phospholipids. Quenching of spectrin intrinsic fluorescence by phospholipid suspensions

Phospholipid suspensions prepared of phosphatidylethanolamine, phosphatidylserine and their mixtures are able to influence the intrinsic protein fluorescence of spectrin. In the case of phosphatidylethanolamine suspension up to 75% of protein fluorescence can be quenched. The interaction of phospholipid aggregates with spectrin is modulated by pH and ionic strength. Phospholipids, particularly phosphatidylethanolamine display a 'stabilizing' effect against the changes of protein fluorescence induced by increasing ionic strength and by thermal denaturation.     

Interaction between microtubule-associated protein tau and spectrin

Tau factor, one of the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography of brain MAPs, gel overlay of electrophoresed MAPs with ¹²⁵I-labelled spectrin, incorporation of tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin.The wide distribution of both tau and spectrin-like proteins in eukaryotic cells in in favor of the possible biological significance of this interaction. The results suggest that tau could be one of the proteins involved in the concerted regulation of microtubule and actin networks in the membrane vicinity.     

Interaction of the hemin 2 and 4 substituents with apo horseradish peroxidase

2-formyl, 4-vinyl deuterohemin (1) and 2-vinyl, 4-formyl deuterohemin (2) substituted horseradish peroxidases have been prepared from apoperoxidase and the respective hemins. The two hemins bind at different rates to the apoprotein and the resultant substituted peroxidases possess different visible spectra and activities. These results indicate that the hemin 2 and 4 substituents interact with apoperoxidase and are not exposed to the solvent.     

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of albumins and hemin with aromatic antioxidants: A spectrophotometric and fluorometric study

Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants(K _a) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4).K _a values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes witho-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media;K _a values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically;K _a values were of order of ∼10⁵ M⁻¹ and decreased with the ligand hydrophobicity.     

Interaction of anilinonaphtyl labeled spectrin with fatty acids and phospholipids: a fluorescence study

Anilinonaphtyl labeled spectrin exhibits a fluorescence emission spectrum characteristic of a highly hydrophobic environment. Quenching of the fluorescence intensity by nitroxide analogs of fatty acids of affinity 10(4) M-1 reveals that the sites of interaction of fatty acids lie very close to the anilinonaphtyl groups. Similar experiments performed with a nitroxide analog of phosphatidylserine yield a 30% quenching of fluorescence while the same phosphatidylcholine analog has essentially no effect. The changes in the fluorescence emission spectrum exhibited in the presence of sonicated phosphatidylserine vesicles further outline the specificity of interaction towards phosphatidylserine, with one spectrin binding site per about 750 exposed phospholipids. Moreover, they suggest a penetration of the anilinonaphtyl group into the lipid bilayer.     

Interaction between the subunits of human erythrocyte spectrin using a fluorescence probe

Fluorescence labeling of spectrin subunits was performed with N-(1-anilinonaphthyl-4)maleimide (ANM) to study the interaction between alpha and beta subunits. The fluorescence anisotropy of both ANM alpha and ANM beta increased linearly with the addition of nonfluorescent beta or alpha subunit, and saturated at a protein ratio about 1, indicating that 1 mol alpha subunit binds to 1 mol beta subunit with high affinity in vitro. Furthermore, this binding seemed to be reversible, because the anisotropy value decreased when an excess fo nonfluorescent alpha was added to the ANM alpha/beta mixture. The anisotropy of ANM alpha attained a maximum level within l min after addition of the same quantity of nonfluorescent beta at 12 degrees C, and the anisotropy of this mixture decreased rapidly when an excess of nonfluorescent alpha was added. These findings suggested that both the binding process of beta to ANM alpha and the dissociation step of ANM alpha from the ANM alpha-beta complex were quite rapid. The results obtained here imply that dynamic interaction between alpha and beta subunits of spectrin should be taken into account in understanding the role of the spectrin molecule in the cytoskeletal mesh.     

Avian lens spectrin: subunit composition compared with erythrocyte and brain spectrin

Chicken lens spectrin is composed predominantly of equimolar amounts of two polypeptides with solubility properties similar, but not identical, to erythrocyte spectrin. The larger polypeptide, Mr 240,000 (lens alpha-spectrin), co-migrates with erythrocyte and brain alpha-spectrin on one- and two-dimensional SDS polyacrylamide gels and cross-reacts with antibodies specific for chicken erythrocyte alpha-spectrin; the smaller polypeptide, Mr 235,000 (lens gamma-spectrin), co-migrates with brain gamma-spectrin and does not cross-react with either the alpha-spectrin antibodies specific for chicken erythrocyte beta-spectrin. Minor amounts of polypeptides antigenically related to erythrocyte beta-spectrin with a greater electrophoretic mobility than lens gamma-spectrin are also detected in lens. The equimolar ratio of lens alpha- and gamma-spectrin is invariantly maintained during the extraction of lens plasma membranes under different conditions, or after immunoprecipitation of whole extracts of lens with erythrocyte alpha-spectrin antibodies. Two-dimensional peptide mapping reveals that whereas alpha-spectrins from chicken erythrocytes, brain, and lens are highly homologous, the gamma-spectrins, although related, have some cell-type-specific peptides and are substantially different from erythrocyte beta-spectrin. Thus, the expression of cell-type-specific gamma- and beta-spectrins may be the basis for the assembly of a spectrin-plasma membrane complex whose molecular composition is tailored to the functional requirements of the particular cell-type.     

Association of spectrin with desmin intermediate filaments

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.     

The interaction of hemin with skeletal muscle actin

The ability of actin to interact with hemin was studied. It was found that the Soret absorption band of hemin changes in the presence of actin and that hemin is capable of quenching the fluorescence intensity of actin. These findings were indicative of hemin binding to actin. The binding constant for the high affinity site was calculated to be 5.3 X 10(6) M-1. The amounts of native G- and F-actin were estimated by their DNAase I inhibition activity. It was observed that the binding of hemin to G-actin is followed by a slow decrease in the ability of actin to inhibit DNAase I activity and to polymerize upon addition of salts. Binding of hemin to F-actin resulted in a gradual depolymerization of the filaments, to an inactivated form, as expressed by a reduction in the ability of hemin-bound F-actin to inhibit DNAase I activity in the absence as well as in the presence of guanidine-HCl. Electron microscopy studies further corroborated these findings by demonstrating that: (1) hemin-bound G-actin failed to show formation of polymers when salts were added; (2) a marked reduction in the amount of actin polymers was observed in the specimens examined 24 h after mixing with hemin. It is suggested that the elevated amounts of free hemin formed under pathological conditions, might be toxic to cells by interfering with actin polymerization cycles.     

Association of iron-protoporphyrin-IX (hemin) with myosins

Addition of myosins isolated from guinea pig heart and rabbit skeletal muscle to hemin solutions resulted in the appearance of new absorption spectra indicating association of hemin and the myosins. Binding stoichiometry based on absorption changes was found to be two hemin sites per myosin molecule. The binding constants calculated from quenching of the intrinsic fluorescence of the myosins by hemin are Ka = 7 (+/- 2) 10(6) M-1 for skeletal muscle myosin, and Ka = 3 (+/- 1) x 10(7) M-1 for heart muscle myosin. Based on these findings, myosins are suggested as potential transporters of free hemin between cell organelles.     

Fractionation of spectrin by differential precipitation with calcium

The calcium-stimulated ATPase activity of spectrin can be substantially separated from the magnesium-stimulated activity by partial precipitation of spectrin with calcium. The precipitated Ca-ATPase fraction can be dissolved by dialysis against EDTA, and the resulting solution can be polymerized into characteristic spectrin fibrils. The supernatant contains Mg-ATPase and, either before or after dialysis, contains predominantly “torus” forms. The two fractions are essentially identical on gel electrophoresis, being composed primarily of the high molecular weight peptides with a constant level of minor bands. These results imply that the Ca-ATPase is associated firmly with the high molecular weight peptides.