Separate sites of low and high affinity for agonists on Torpedo californica acetylcholine receptor

We have studied alkylation of the membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electric organ by the cholinergic agonist bromo-acetylcholine (BrAcCh). Following reduction of the AcChR with dithiothreitol (DTT) under strictly controlled conditions, a single class of binding sites was covalently labeled by BrAcCh. The extent of alkylation was dependent on the concentration of both DTT and BrAcCh and reached a maximum when a number of sites equivalent to the number of alpha-bungarotoxin (alpha-BTx) binding sites were labeled. The reaction with BrAcCh was completely inhibited by saturating concentrations of alpha-BTx. On the contrary, complete alkylation of the AcChR with [3H]BrAcCh consistently inhibited only approximately 50% of alpha-BTx binding. The effects of DTT reduction and subsequent BrAcCh alkylation on the cation-gating properties of the AcChR were investigated in rapid kinetic experiments. DTT reduction resulted in a slight decrease in the maximum cation flux and a small shift in the effective dissociation constant to higher acetylcholine (AcCh) concentration. The flux response was completely inhibited by maximal alkylation of the membrane vesicles by BrAcCh. A low-affinity binding site for AcCh, which is likely to be important in AcChR activation, has been revealed for T. californica AcChR by studying the effects of cholinergic ligands on the fluorescence of a probe, 4-[(iodoacetoxy)ethylmethylamino]-7-nitro-2,1,3-benzoxadiazole (IANBD), covalently bound to the AcChR protein. Maximal labeling by BrAcCh did not affect the binding of AcCh to the low-affinity binding site, as monitored by changes in the fluorescence of this probe. This low-affinity binding site must therefore be distinct from the site labeled by BrAcCh. The results strongly support the notion that the nicotinic AcChR contains multiple binding sites for cholinergic ligands.     

Binding of anti-acetylcholine receptor antibodies inhibits the acetylcholine receptor mediated cation flux

A fast kinetics, spectroscopic technique has been applied to the study of the transient cation flux associated to the binding of cholinergic agonist to native acetylcholine receptor (AcChR)-rich membrane vesicles in presence of anti-AcChR antibodies. The technique is based on the collisional quenching of an intravesicularly trapped fluorophore by externally added T1+ which substitutes for physiologically occurring cations. Presence of polyclonal Fab fragments from goat anti-AcChR antibodies bound to the membrane AcChR promotes a 80-90% inhibition on the observed rate constants of T1+ influx. The observed inhibition process appears to follow a non-competitive pattern between antibody and cholinergic ligand binding, suggesting that in the AcChR protein the antigenic sites responsible for ion translocation may be other than those involved in ligand binding.     

Fourier-transform infrared spectroscopy study of the pressure-induced changes in the structure of the bovine alpha-lactalbumin: the stabilizing role of the calcium ion.

The Fourier-transform infrared spectroscopy (FTIR) technique with a diamond anvil cell has been applied for examination of the pressure-induced changes occurring in the secondary structure of the alpha-lactalbumin. This is the first high-pressure FTIR study of a calcium-binding protein which simultaneously takes into account spectral changes in both the calcium-ion-binding carboxyl groups' band and the amide I/I' vibrational band. Spectral behavior of three kinds of the protein: the undeuterated holoform, the fully deuterated holoform, and the undeuterated apoform was compared in the pressure range from 0.1 MPa up to 740 MPa. We found that the binding of calcium remarkably stabilizes the alpha-lactalbumin against pressure as it is followed approximately by a 200-MPa increase of the value of pressure at which denaturation occurs. A quantitative analysis of the band of antisymmetrical stretching vibrations of the calcium-binding carboxyl groups revealed that the pressure-induced changes in the calcium-binding loop occur in two stages. Binding of the calcium ion seemingly increases the pressure-stability of the calcium-binding loop to a higher degree than the pressure-stability of the secondary structure of the alpha-lactalbumin. We have also discussed in detail the complex pressure-enhanced H/D exchange in the alpha-lactalbumin. Finally, we have proposed a new assignment of major peaks in the helical region of the amide I/I' spectral band of the partially deuterated alpha-lactalbumin.     

Monoclonal antibodies as probes of the alpha-bungarotoxin and cholinergic binding regions of the acetylcholine receptor

We have probed the acetylcholine receptor (AcChR) molecule with six anti-AcChR monoclonal antibodies (mAbs) whose binding to the AcChR is inhibited or blocked by alpha-bungarotoxin (alpha BgTx). mAbs bound with a maximum stoichiometry of either one mAb (387D, 247G) or two mAbs (383C, 572C, 370C, 249E) per AcChR monomer, and the extent to which they inhibited alpha BgTx binding directly correlated with their stoichiometry of binding. The effect of mAbs on the alpha BgTx and cholinergic ligand binding properties of the AcChR molecule defined three major categories of mAbs: those that block alpha BgTx and carbamylcholine (agonist) binding, but do not block d-tubocurarine (antagonist) binding (383C, 572C, 370C and 249E); mAb 387D, which blocks agonist binding and partially blocks alpha BgTx and d-tubocurarine binding; and mAb 247G, which does not affect agonist binding, blocks at most 50% of the alpha BgTx binding sites, and decreases the affinity of the high affinity component of d-tubocurarine binding (Mihovilovic, M., and Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059). Except for mAb 247G, these mAbs strongly competed with each other for binding to the AcChR. In contrast, mAb 247G blocks about 50% of the binding of all the other mAbs. The results demonstrate the ability of mAbs to stabilize different conformational states of the AcChR and to probe cholinergic epitopes of functional importance. They also indicate the nonequivalence of the two alpha-toxin binding regions of the AcChR molecule and suggest that it is possible to identify epitopes within the alpha BgTx binding region that when bound produce differential effects on the binding of the agonist (carbamylcholine) and the antagonist (d-tubocurarine).     

Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells

We report the first Fourier transform infrared analysis of prion protein (PrP) repeats and the first study of PrP repeats of marsupial origin. Large changes in the secondary structure and an increase in hydrogen bonding within the peptide groups were evident from a red shift of the amide I band by >7 cm(-1) and an approximately five-fold reduction in amide hydrogen-deuterium exchange for peptide interacting with Cu(2+) ions. Changes in the tertiary structure upon copper binding were also evident from the appearance of a new band at 1564 cm(-1), which arises from the ring vibration of histidine. The copper-induced conformational change is pH dependent, and occurs at pH >7.     

Investigation of adenylate kinase from Escherichia coli and its interaction with nucleotides by Fourier transform infrared spectroscopy

The secondary structure of adenylate kinase (EC 2.7.4.3) from E. coli was investigated under various conditions using Fourier transform infrared spectroscopy. The overall band contour of the conformation-sensitive amide I mode indicates that in HEPES buffer (pH 7.4) the major structure of the protein is alpha-helical. A more detailed estimate obtained from decomposition of the amide I band into its constituent component bands gives 50% alpha-helix, 26% beta-structure, 15% turns and loops, and about 9% nonrepetitive unordered structures. Binding of nucleotide (e.g., ATP) to the donor site decreases the beta-content and shifts the amide I band to higher wavenumbers, whereas binding of nucleotide (e.g., AMP) to the acceptor site does not produce any change in conformation of the protein. These results agree with the protection by ATP and lack of protection by AMP when adenylate kinase is digested with trypsin. The effect of protein denaturing agents and conditions (temperature, high pH, sodium dodecyl sulfate) on changes in the protein conformation as revealed by the conformation-sensitive amide I bands is discussed.     

Pyrenesulfonyl azide as a fluorescent label for the study of protein-lipid boundaries of acetylcholine receptors in membranes

Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core. Covalent attachment of the photogenerated fluorescence probe does not perturb the α-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra or fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.     

Protective effects of agmatine in rotenone-induced damage of human SH-SY5Y neuroblastoma cells: fourier transform infrared spectroscopy analysis in a model of Parkinson's disease

Agmatine is a novel neuromodulator that plays a protective role in the CNS in several models of cellular damage. However, the mechanisms involved in these protective effects in neurodegenerative diseases are poorly understood. Fourier transform infrared (FTIR) spectroscopy analysis detects biomolecular changes in disordered cells and tissues. In this report, we utilize FTIR spectroscopy to characterize the changes in rotenone-induced damage in neuronal-like differentiated SH-SY5Y neuroblastoma cells in the presence or absence of agmatine. The analysis of the FTIR spectra demonstrates significant alterations in rotenone-treated cells, whereas the FTIR spectra obtained after pre-incubation with agmatine (250 nM) significantly reduces these redox alterations and more closely resembles those of the control cells. In particular, rotenone-damaged cells demonstrate spectral alterations related to amide I, which correspond to an increase in β-sheet components, and decreases in the amide II absorption intensity, suggesting a loss of N-H bending and C-N stretching. These alterations were also evident by Fourier self-deconvolution analysis. Thus, rotenone-induced increases in the levels of stretching vibration band related to the protein carboxyl group would account for a significant amount of misfolded proteins in the cell. Agmatine effectively reduces these effects of rotenone on protein structure. In conclusion, antioxidant and scavenging properties of agmatine reduce rotenone-produced cellular damage at the level of protein structure. These, together with other previous observations, demonstrate the therapeutic potential of agmatine in the treatment of Parkinson's disease.     

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.     

Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.     

Redox-dependent changes in beta-extended chain and turn structures of cytochrome c in water solution determined by second derivative amide I infrared spectra.

The redox-dependent changes in secondary structure of cytochromes c from horse, cow, and dog hearts in water at 20 degrees C have been determined by amide I infrared spectroscopy. Second derivative amide I spectra were obtained by use of a procedure that includes a convenient method for the effective subtraction of the spectrum of water vapor in the system. The band at 1657 cm-1 representing the helix structure was unaffected by a change in redox state whereas changes in bands due to turns at 1680, 1672, and 1666 cm-1, unordered structure at 1650 cm-1, and beta-structures at 1632 and 1627 cm-1 occurred. About one-fourth of the beta-extended chain spectral region and one-fifth of the beta-turn region (involving a total of approximately 9-13 residues) were sensitive to the oxidation state of heme iron. No significant changes in the secondary structure of either the reduced or oxidized protein due to changes in ionic strength were detected. The localized structural rearrangements triggered by the changes in oxidation state of heme iron are consistent with differences in the binding of heme iron to a histidine imidazole nitrogen and a methionine sulfur atom from the beta-extended chain. The demonstrated ability to obtain highly reproducible second derivative amide I infrared spectra confirms the unique utility of such spectral measurements for localization of subtle changes in secondary structure within a protein, especially for changes among the multiple turns and beta-structures.     

The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.     

Investigation of secondary and tertiary structural changes of cytochrome c in complexes with anionic lipids using amide hydrogen exchange measurements: an FTIR study.

The structure of cytochrome c bound to anionic lipid membranes composed of dimyristoyl, dipalmitoyl, or dioleoyl phosphatidylglycerols, or of bovine heart cardiolipin, has been investigated by Fourier transform infrared spectroscopy. Only small changes in secondary structure, as registered by the amide I band of cytochrome c, were observed upon binding at temperatures below that of denaturation of the protein, and these were not coupled to the thermotropic phase transitions of the lipid. The denaturation temperature of the protein decreased by approximately 25-30 degrees upon binding, in a progression which correlated with that of the lipid phase transition temperatures, being approximately 7 degrees lower for complexes with dioleoyl than with dipalmitoyl phosphatidylglycerol. Large changes in the amide proton exchange characteristics, as monitored by the spectral shifts in the amide I band of the protein in D2O, were observed on binding cytochrome c to the lipid membranes. For the slowly exchanging population, the amide deuteration rates of the free protein were nearly independent of temperature, whereas those of the bound protein increased by up to two orders of magnitude over the temperature range from 10 to 40 degrees C. In addition, the extent of exchange differed between the bound and unbound protein. A structural transition in the bound protein was detected as a discontinuous step in Arrhenius plots of the deuterium exchange rates which occurred at a temperature in the region of 22 to 29 degrees C, depending on the lipid, far below that of denaturation. The temperature of this transition was determined by the physical state of the lipid, being 7 degrees lower for the lipids in the fluid state than for those in the gel state, and, for complexes with dimyristoyl phosphatidylglycerol, occurred at an intermediate temperature, being controlled by the lipid chain-melting transition at 27-28 degrees C. These results provide evidence for a coupling of the tertiary structure of the membrane-bound protein with the physical state of the membrane lipids.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

Generation of a substructure library for the description and classification of protein secondary structure. II. Application to spectra-structure correlations in Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has become well known as a sensitive and informative tool for studying secondary structure in proteins. Present analysis of the conformation-sensitive amide I region in protein infrared spectra, when combined with band narrowing techniques, provides more information concerning protein secondary structure than can be meaningfully interpreted. This is due in part to limited models for secondary structure. Using the algorithm described in the previous paper of this series, we have generated a library of substructures for several trypsin-like serine proteases. This library was used as a basis for spectra-structure correlations with infrared spectra in the amide I' region, for five homologous proteins for which spectra were collected. Use of the substructure library has allowed correlations not previously possible with template-based methods of protein conformational analysis.     

Thermal perturbation studies of membrane-bound acetylcholine receptor from Torpedo: effects of cholinergic ligands and membrane perturbants

Thermal perturbation techniques have been used to probe structural features of the nicotinic acetylcholine receptor (AcChR). The information obtained from differential scanning calorimetry (DSC) of AcChR membranes (M.C. Farach and M. Martinez-Carrion (1983) J. Biol. Chem. 258, 4176) in the absence and in the presence of cholinergic ligands and local anesthetics, is comparable to that obtained from a simpler technique of heat inactivation of the alpha-bungarotoxin (alpha-Bgt) binding sites on the AcChR protein in similar samples. When AcChR membranes are heated at approximately 1 degree C/min, heat inactivation of toxin binding sites has a characteristic T50 value (temperature at which 50% of the initial capacity to bind alpha-Bgt remains) of approximately 60 degrees C. When heated at a constant temperature during increasing periods of time, the rate at which heat inactivation occurs is also characteristic of the temperature chosen for the experiment. The above thermal parameters are also sensitive to perturbation of the AcChR membrane matrix by the presence of subsolubilizing concentrations of detergents. Moreover, elimination of detergents by dialysis allows us to evaluate the reversibility or irreversibility of AcChR thermal destabilization induced by detergents or other membrane perturbants. Under the experimental conditions used, structural destabilization induced by octylglucoside or cholate can be fully reversed by detergent dialysis, while that exerted by deoxycholate cannot. "Thermal gel" analysis of the aggregation of AcChR subunits induced by heat (G. Soler, J. R. Mattingly, and M. Martinez-Carrion (1984) Biochemistry 23, 4630) has also been used to assess the effects of detergent presence on the AcChR protein. When deoxycholate is used as the perturbing agent, there is a particularly effective sulfhydryl-mediated aggregation of the gamma-delta subunit group, which appears to correlate with the irreversible destabilization of alpha-Bgt binding sites induced by that detergent.     

Structure and chemical modifications of neurotoxin from Naja nigricollis studied by Raman spectroscopy

Raman spectroscopy was used to determine structural features of the native toxin alpha from Naja nigricollis, which contains only one Trp and one Tyr, and of chemically modified toxins having chromophores added to these two conserved aromatic amino acids. The percentages of secondary structure were determined by using amide I polypeptidic vibration analysis and are in agreement with X-ray structure [Low et al. (1976) Proc. Natl. Acad Sci. U.S.A. 73, 2991-2994] as well as with the geometry of the disulfide bridges estimated by using the v(S-S) vibrations. In the native toxin alpha, the single invariant tyrosine 25 appears to be buried in the structure and involved in a strong hydrogen bond. We have chemically modified these two invariant aromatic side chains by addition of chromophores. The presence of a (nitrophenyl)sulfenyl (NPS) chromophore bound to the Trp does not perturb the secondary structure of the toxin as shown by the analysis of the polypeptidic amide I vibrations; however, the environment of this Trp and the geometry of a disulfide bridge seem to be modified. The secondary structure is not affected by the presence of the NPS chromophore; therefore, the decrease in binding affinity observed after modification of Trp-29 by the reagent NPS-Cl [Faure et al. (1983) Biochemistry 22, 2068-2076] is due to an alteration of the environment of this aromatic amino acid and/or a steric hindrance and not to an overall modification of the toxin structure. The binding assays of [nitrotyrosyl]toxin show that after nitration the affinity toward the monoclonal antibody M alpha 1 is unchanged and that the affinity toward the cholinergic receptor (AcChR) from Torpedo marmorata remains high. We concluded that the structure of toxin alpha after adding the NO2 chromophore to Tyr-25 is the same as it is in native toxin.     

Structure and function of an acetylcholine receptor.

Structural analysis of an acetylcholine receptor from Torpedo californica leads to a three-dimensional model in which a "monomeric" receptor is shown to contain subunits arranged around a central ionophoretic channel, which in turn traverses the entire 110 A length of the molecule. The receptor extends approximately 15 A on the cytoplasmic side, 55 A on the synaptic side of the membrane. The alpha-bungarotoxin/agonist binding site is found to be approximately 55 A from the entrance to the central gated ion channel. A hypothesis for the mechanism of AcChR is presented which takes into account the structural and kinetic data, which is testable, and which serves as a focus for future studies on the agonist-induced structure change in AcChR.     

Interaction between hesperetin and human serum albumin revealed by spectroscopic methods

Hesperetin (5,7,3'-trihydroxyl-4'-methoxyl-flavanone) is an important bioactive compound in Chinese traditional medicine and has multiple biological and pharmacological activities. The interaction of hesperetin with human serum albumin (HSA) has been investigated by UV absorption, fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of drug at the molar ratio of drug to HSA ranging from 0.3 to 7 and the binding affinity (K(A)) was 8.11x10(4) M(-1). The primary binding site was most likely located on subdomain IIA. The binding ability of the drug to protein decreased from pH 6.4 to 8.4 in the drug to protein molar ratio of 1. Combining the curve-fitting results of infrared amide I band in D2O and H2O phosphate buffers, the alterations of protein secondary structure after drug complexation were estimated. With increasing the drug concentration, the percentage of protein alpha-helix structure decreased gradually. The reduction of protein alpha-helix structure reached about 7-9% after the protein interacted with hesperetin in D2O and H2O buffer solution at pH 7.4 when the drug to protein molar ratio was 10. This indicated a partial unfolding of HSA in the presence of the drug. From the results of UV absorption, fluorescence and Fourier transformed infrared spectrometry, the binding mode was discussed. The main mechanism of protein fluorescence quenching was a static quenching process and the hydroxyl groups of the drug in its neutral part played an important role in the binding process.     

Interactions of human serum albumin with chlorogenic acid and ferulic acid

The interactions of chlorogenic acid and ferulic acid with human serum albumin (HSA) have been investigated by fluorescence and Fourier transformed infrared (FT-IR) spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of drugs at the molar ratio of drug to HSA ranging from 1 to 10, and their binding affinities (KA) are 4.37 x 10(4) M(-1) and 2.23 x 10(4) M(-1) for chlorogenic acid and ferulic acid, respectively. The primary binding site for chlorogenic acid is most likely located on IIA and that for ferulic acid in IIIA. The main mechanism of protein fluorescence quenching was static quenching process. Combining the curve-fitting results of infrared amide I and amide III bands, the alterations of protein secondary structure after drug complexation were estimated. With increasing the drug concentration, the protein alpha-helix structure decreased gradually and the reduction of protein alpha-helix structure reached about 7% and 5% for protein binding with chlorogenic acid and ferulic acid individually at the drug to protein molar ratio of 30. This indicated a partial unfolding of HSA in the presence of the two acids. From the fluorescence and FT-IR results, the binding mode was discussed.