The acid-induced state of glucose oxidase exists as a compact folded intermediate

A systematic investigation of the acid-induced unfolding of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase) (GOD) from Aspergillus niger was made using steady-state tryptophan fluorescence, circular dichroism (CD), and ANS (1-anilino 8-naphthalene sulfonic acid) binding. Intrinsic tryptophan fluorescence studies showed a maximally unfolded state at pH 2.6 and the presence of a non-native intermediate in the vicinity of pH 1.4. Flavin adenine dinucleotide (FAD) fluorescence measurements indicate that the bound cofactors are released at low pH. In the pH range studied, near- and far-UV CD spectra show maximal loss of tertiary as well as secondary structure (40%) at pH 2.6 although glucose oxidase at this pH is relatively less denatured as compared to the conformation in 6M GdnHCl. Interestingly, in the vicinity of pH 1.4, glucose oxidase shows a refolded conformation (A-state) with approximately 90% of native secondary structure and native-like near-UV CD spectral features. ANS fluorescence studies, however, show maximal binding of the dye to the protein at pH 1.4, indicating a "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a more compact conformation at low pH. Thermal stability of this state was assessed by ellipticity changes at 222 nm relative to native protein. While native glucose oxidase showed a completely reversible thermal denaturation profile, the state at pH 1.4 showed approximately 50% structural loss and the denatured state appeared to be in a different conformation exhibiting prominent beta-sheet structure (around 85 degrees C) that was not reversible. To summarize; the A-state of GOD exists as a compact folded intermediate with "molten-globule"-like characteristics, viz., native-like secondary structure but with non-native cofactor environment, enhanced hydrophobic surface area and non-cooperative thermal unfolding. That the A-state also possesses significant tertiary structure is an interesting observation made in this study.     

Evaluation of binding and thermodynamic characteristics of interactions between a citrus flavonoid hesperitin with protein and effects of metal ions on binding

The mechanism of interaction of a non-glycosidic citrus flavonoid, hesperitin (HES) with bovine serum albumin (BSA) was studied by UV-vis absorption, fluorescence, FT-IR, circular dichroism, fluorescence anisotropy and synchronous fluorescence spectroscopy in phosphate buffer of pH 7.4. Fluorescence data revealed that the fluorescence quenching of BSA by HES was the result of the formed complex of HES-BSA. The binding constants and thermodynamic parameters at four different temperatures, the location of binding, and the nature of binding force were determined. The hydrogen bonds interactions were found to be the predominant intermolecular forces to stabilize the complex. The conformation of BSA was discussed by synchronous fluorescence and CD methods. The alterations of protein secondary structure upon complexation with HES were evident from the gradual decrease in α-helicity. The distance between the donor (BSA) and acceptor (flavonoid) was calculated from the fluorescence resonance energy transfer and found to be 1.978 nm. Common ions viz., Zn(2+), K(+), Cu(2+), Ni(2+), Mn(2+) and Co(2+) were found to influence the binding of flavonoid to protein.     

Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.     

Spectral studies of human erythrocyte catalase.

The optical absorption and circular dichroic spectra of human erythrocyte catalase (EC 1.11.1.6) and its cyanide, azide, and fluoride derivatives over the wavelength range of 210 to 700 nm are reported. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the heme group from the protein. The fractions of the protein structure present as alpha helix, beta pleated sheet, and unordered structure have been estimated from the CD spectrum in the far-ultraviolet region. The CD spectra also indicate that the protein conformation does not change appreciably after cyanide binding. The epr spectroscopy of the native enzyme and its cyanide complex are reported. The spectral results are compared with catalase obtained from other mammalian and bacterial sources.     

Spectroscopic characterization of Phaseolus vulgaris leucoagglutinin

Phaseolus vulgaris leucoagglutinin is a homotetrameric legume lectin possessing the canonical dimeric structure common to other legume lectins. In order to gain insight into the stability of the protein in an acidic environment, it was characterized by CD and fluorescence studies at pH 2.5. This was then compared with the native protein at physiological pH (7.2). The extinction coefficient of the native protein was calculated to be 3.58x10(4) from its UV absorption spectra. The far- and near-UV CD spectra of the protein at pH 2.5 showed very little difference even though the protein was found to exist as a dimer at pH 2.5. The fluorescence emission maxima of the protein upon excitation at 280 nm were found to shift only from 331 nm at pH 7.2 to 333 nm at pH 2.5. Based on the above observation it was concluded that the protein exhibits extreme pH stability especially in the acidic range. The secondary and tertiary structure of the protein is lost only when it is incubated for two days in 6 M GdnHCl at pH 2.5. At pH 7.2 it could be denatured in 6 M GdnHCl after one week of incubation.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.     

A study of flavin-protein and flavoprotein-ligand interactions. Binding aspects and spectral properties of D-amino acid oxidase and riboflavin binding protein.

To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein(RBP),D-amino acid oxidase(D-AO) and its complexes with ligands were observed in the spectral region of 310-600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 x 10(-8)M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein-ligand interaction. Comparison of the absorption, CD and MCD spectra(310-600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.     

Phage T4 lysozyme. Physical properties and reversible unfolding.

Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07-1 g/l the c-m equals 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c-m equals 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10-4 M dithiothreitol and 10-6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T-m, increment H-unf and increment S-unf with increasing pH.     

The interaction of blood proteins with brucine

The features of brucine (BC) binding to two blood proteins, bovine hemoglobin (BHb), and bovine serum albumin (BSA), were investigated via fluorescence, circular dichroism and UV/Vis absorption spectroscopy. The results revealed that BC caused the fluorescence quenching of blood proteins by the formation of BC–protein complex. The corresponding thermodynamic parameters were measured at different temperatures. The process of binding BC molecule on protein was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The molecular docking has been employed to explore the binding site of the BC in BHb and BSA on the Autodock 4.2. The distances r between BC and protein were calculated to be 4.93 and 5.08 nm for BHb, and BSA, respectively. The effect of BC on the conformation of blood proteins was analyzed using CD, synchronous fluorescence and three-dimensional fluorescence spectra.     

The protein conformation and a zinc-binding domain of an autoantigen from mouse seminal vesicle.

The protein conformation of a mouse seminal vesicle autoantigen was studied by circular dichroism spectroscopy. At pH 7.4, the spectrum in the UV region appears as one negative band at 217 nm and one positive band at 200 nm. This together with the predicted secondary structures indicates no helices but a mixture of beta form, beta turn, and unordered form in the protein molecule. The conformation is stable even at pH 10.5 or 3.0. The spectrum in the near-UV region consists of fine structures that are disturbed in acidic or alkaline solution. The environments around Trp2 and Trp82 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 345 nm, and about 87% of them are accessible to quenching by acrylamide. Correlating the quenching effect of CsCl and Kl on the protein fluorescence to the charged groups along the polypeptide chain suggests the difference in the "local charge" around the two tryptophan residues. The presence of ZnCl2 in the protein solution effects no change in the circular dichroism but perturbs the fluorescence due to Trp82. Analysis of the fluorescence data suggests a Zn(2+)-binding site on the protein, which cannot coordinate with both Ca2+ and Mg2+. The association constant for the complex formation is 1.35 x 10(5) +/- 0.04 x 10(5) M-1 at pH 7.4.     

Understanding bilirubin conformation and binding. Circular dichroism of human serum albumin complexes with bilirubin and its esters

Human serum albumin binds tightly and noncovalently to a wide variety of hydrophobic bilirubins, including (4Z,15Z)-bilirubin-IX alpha, its dimethyl ester and mono methyl esters, its mono 2-butyl esters and amides, the dimethyl ester of (4Z,15Z)-mesobilirubin-IV alpha, and even (4Z,15Z)-etiobilirubin-IV gamma. The heteroassociation complexes formed from these highly water-insoluble pigments and the protein can be prepared in pH 7.4 aqueous by using a small quantity of dimethyl sulfoxide as amphiphilic carrier. In those solutions the protein acts as a water-soluble chiral complexation agent to induce an asymmetric transformation of the bound pigment. This is recognized by positive chirality, bisignate induced circular dichroism (CD) Cotton effects that fall in the region of the bichromophoric pigment's long wavelength UV-visible absorption band and are characteristic of intramolecular exciton coupling of the bilirubin component pyrromethenone chromophores. The same-signed CD spectra shared by all the pigments of this work indicate selection at the protein binding site for a positive chirality conformer and suggest a common binding site. The CD intensities, which are greatest ([delta epsilon[ congruent to 50) for pigments with one or two free carboxyl groups, are consistent with a binding model where one salt linkage plays a major role in the enantioselectivity of the right-handed folded conformation stabilized by inter- and intramolecular hydrogen bonds.     

Effect of metal ions and EGTA on the optical properties of concanavalin A at alkaline pH

In our earlier communications, we reported the effect of salts and alcohols on alpha-chymotrypsinogen [1] and the existence of stable intermediates at low pH in bromelain [2] and glucose oxidase [3]. In the present study, the role of metal ions and EGTA on the conformation of concanavalin A at alkaline pH was studied by near- and far-UV circular dichroism, fluorescence emission spectroscopy and binding of a hydrophobic dye, 1-anilino-8-naphthalene sulfonate (ANS). Far-UV CD spectra showed the transition from an ordered secondary structure at pH 7 with a trough at 223 nm to a relatively unordered state at pH 12. Near-UV CD spectra showed the loss of signal at 290 nm, thereby indicating the disruption of native three dimensional structure. Maximum ANS binding occurred at pH 12 suggesting the presence of an intermediate or molten globule-like state at alkaline pH.     

Conformational States of Trifluoroacetic Acid–Treated Cytochrome c in the Presence of Salts and Alcohols

We have carried out a systematic investigation of salts- and alcohols-induced conformational alterations on the trifluoroacetic acid (TFA)-treated ferricytochrome c by soret absorption spectroscopy, far UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-8-naphthalene sulfonate (ANS) binding. TFA induces the unfolding of native cytochrome c obtained from horse heart leading to loss of secondary structure. The addition of increasing concentration of salts and alcohols leads to increase in MRE value at 222 and 208 nm indicating an increase in the alpha-helical content leading to formation of compact dimensional structure. Cytochrome c is a heme protein in which the resonance energy of tryptophan is transferred to heme resulting in quenched tryptophan fluorescence. Addition of alcohols leads to increase in tryptophan and ANS fluorescence. The tryptophan and ANS fluorescence in case of salts shows decreased fluorescence intensity. TFA-induced unfolded cytochrome c showed the soret absorption maximum at 394 nm. However, an intermediate state in presence of alcohols and salts showed the absorption maxima at 398 nm and 402 nm, respectively. Among all the salts and alcohols studied, K3Fe(CN)6 and butanol were found to be most effective as examined by the above-mentioned spectroscopic techniques. The order of effectiveness of alcohols was found to be butanol > propanol > ethanol > methanol. The following effective trend in the case of salts was obtained: K3Fe(CN)6 > K2SO4>KClO4 > KCl. These results suggest that alcohols induce an intermediate with molten globule-like conformation on the TFA unfolded state, whereas salts induce a refolded intermediate approaching native-like conformation.     

Circular dichroic and fluorometric studies on the acid-induced isomerization of bovine plasma albumin--1 -anilino-8-naphthalenesulfonate complex

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin--1-anilino-8-naphthalenesulfonate complex, BPA-ANS1.0 complex (molar ratio of added ANS to BPA = 1.0) were studied by measuring fluorescence and induced CD spectra of ANS. Decrease in the reciprocal of fluorescence polarization, increase in fluorescence intensity and blue shift of fluorescence of ANS in BPA-ANS1.0 complex were correlated with the initial part of the N-F transition and/or the N-F1 transition. Induced CD spectra of ANS showed positive bands at 250-258 and 320-350 nm and one negative band at 280 nm. Most of changes (decreases) in -[theta]280 were also correlated with the initial part of the N-F transition and/or the N-F1 transition. Changes in fluorescence parameters and induced CD spectra of ANS (-[theta]280) might indicate the conformational changes around a strong ANS binding site in the N-terminal domain (Reed et al. (1975), Jonas & Weber (1970) and Brown & Shockley (1982].     

Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution

The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general.     

Conformational and binding properties of chicken liver basic fatty acid binding protein in solution

The conformation of basic fatty acid binding protein from chicken liver and the binding properties of the apo protein toward 11-dansylamino-undecanoic acid were investigated by CD and fluorescence spectroscopy. In one set of experiments the binding process was followed by the appearance of induced optical activity in the absorption region of the dansyl chromophore. In a second set of experiments the binding process was followed by the large enhancement of emission fluorescence of the dansyl fluorophore. From the saturation curves, the stoichiometry of the complex and the binding constant of the fatty acid to the protein were precisely determined. The values of the dissociation constant determined with the two methods were in excellent agreement: we obtained K_D = (1.0 ± 0.1) · 10^?6M in a 0.9 : 1 stoichiometry. The native conformation of the protein is remarkably stable in a variety of solvent systems, including acetonitrile–water, ethylene glycol–water, and dicxane–water of various compositions. The CD results also showed that the binding of the fatty acid does not induce any appreciable change in the protein conformation. In a mixture of water and 2,2,2-trifluoroethanol 1 : 9 (v/v), the native conformation collapses and a new ordered structure is formed, characterized by a high amount of α-helix. © 1994 John Wiley & Sons, Inc.     

Synthesis of Morin–Zinc(II) Complex and its Interaction with Serum Albumin

A morin–zinc(II) complex (MZ) was synthesized and its interaction with bovine serum albumin (BSA) were studied by molecular spectroscopy including fluorescence emission spectra, UV-visible spectra, circular dichroism (CD) spectra, three-dimensional fluorescence spectra, and synchronous fluorescence spectra. The interaction mechanism of BSA and MZ was discussed by fluorescence quenching method and Förster non-radiation energy transfer theory. The thermodynamic parameters ΔH ^θ, ΔG ^θ, ΔS ^θ at different temperatures were calculated and the results indicate the interaction is an exothermic as well as entropy-driven process. Hydrogen bond forces played the most important role in the reaction. The fluorescence probe experiment showed that the binding site of MZ is in subdomain IIA of BSA and the distance between BSA and MZ is 3.17 nm at normal body temperature. The conformation changes of BSA in presence of MZ were investigated by CD spectra and three-dimensional fluorescence spectra.     

The pH dependence of cytochrome a conformation in cytochrome c oxidase.

The pH dependence of the conformation of cytochrome a in bovine cytochrome c oxidase has been studied by second derivative absorption spectroscopy. At neutral pH, the second derivative spectra of the cyanide-inhibited fully reduced and mixed valence enzyme display two Soret electronic transitions, at 443 and 451 nm, associated with cytochrome a. As the pH is lowered these two bands collapse into a single transition at approximately 444 nm. pH titration of the cyanide-inhibited mixed valence enzyme suggests that the transition from the two-band to one-band spectrum obeys the Henderson Hasselbalch relationship for a single protonation event with a transition pKa of 6.6 +/- 0.1. No pH dependence is observed for the spectra of the fully reduced unliganded or CO-inhibited enzyme. Tryptophan fluorescence spectra of the enzyme indicate that no major disruption of protein structure occurs in the pH range 5.5-8.5 used in this study. Resonance Raman spectroscopy indicates that the cytochrome a3 chromophore remains in its ferric, cyanide-bound form in the mixed valence enzyme throughout the pH range used here. These data indicate that the transition observed by second derivative spectroscopy is not due simply to pH-induced protein denaturation or disruption of the cytochrome a3 iron-CN bond. The pH dependence observed here is in good agreement with those observed earlier for the midpoint reduction potential of cytochrome a and for the conformational transition associated with energy transduction in the proton pumping model of Malmstr?m (Malmstr?m, B. G. (1990) Arch. Biochem. Biophys. 280, 233-241). These results are discussed in terms of a model for allosteric communication between cytochrome a and the binuclear ligand binding center of the enzyme that is mediated by ionization of a single group within the protein.     

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant. The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm. CD spectral analyses with three different methods (S.W. Provencher, J. Gl?ckner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y. Yang, C.-S.C. Wu, H.Z. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208-269; N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet. Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG. These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.