Spectroscopic Probes of the Individual and Combined Effects of Triton X‐100 and Chloroform on Serum Albumins and Serum‐Albumin · Bilirubin Complexes

Abstract

The effects of the non-ionic surfactant Triton X-100 on the biphasic induced CD spectra of bilirubin complexes of human and bovine serum albumins (HSA and BSA) are divergent. While Triton X-100 inverts the induced CD spectrum of HSA . bilirubin, this surfactant enhances the ellipticity values of induced CD of BSA . bilirubin without inversion. The effect of Triton X-100 on the characteristic ultraviolet-CD spectra of the albumins are similar; both the albumins are denatured from their native globular structures. The anionic surfactant SDS, unlike non-ionic Triton X-100, dislodges the ligand from its protein complexes, indicating that both electrostatic and hydrophobic forces are involved in binding of bilirubin to the albumins.The aprotic solvent chloroform inverts the biphasic induced CD spectra of HSA . bilirubin and BSA . bilirubin, whereas CHCI, has relatively little effect on the ultraviolet CD spectra of the albumins. The combined effect of Triton X-I00 and CHCI, shows that the effect of CHCI, predominates over that of Triton X-100. The perturbing effects of Triton X-100 and CHCI, on the CD or induced CD spectra of the proteins or their bilirubin complexes are reversible, and independent of the order in which components were added. The observations suggest that the denaturation of the albumins by Triton X-100 or solvation of CHCI, within albumins markedly alter the internal topography or dynamics of the receptor sites, triggering alterations of the chirality of the bound pigment in sign and/or magnitude.Keywords : bilirubin ; serum albumin ; induced circular dichroism ; enantioselection ; denaturation.Bilirubin, the end product of heme metabolism, is the cytotoxic and neurotoxic pigment of jaundice (Rimington, 1957 ;With, 1968; Brodersen, 1979a;Semba and Kato, 1982;Meisel and Jahr, 1991 ; Ostrow, 1993, 1996;Ostrow et al., 1994;Hansen, 1994). The molecule is a substituted tetrapyrrole dicarboxylic acid. It consists of two dipyrrole halves joined by CH, group. The two dipyrrole halves are intramolecularly hydrogen bonded, between the COOH group of each half and the lactam (NH-C=O) and NH groups of the other half, imposing folded biplanar ridge-tile interconverting enantiomeric conformations (Bonnett et al., 1976(Bonnett et al., , 1978Kaplan and Navon, 1983;Trull et al., 1983Trull et al., , 1987Person et al., 1994;Nogles and Lightner, 1995).Due to the presence of aromatic domains (rings) and imposed conformational restriction, the molecule is hydrophobic in nature, which is unusual for a dicarboxylic acid (Bonnett et al., 1976(Bonnett et al., , 1978 Brodersen, 197913; Lightner and McDonagh, 1984;Lightner et al., 1996). In plasma the pigment is rendered soluble on binding to albumin, the most abundant serum protein, forming a non-covalent-association complex, and is transported to the liver for glucuronidation and subsequent excretion (Ostrow et al., 1963;Cooke and Roberts, 1969; Watson, 1962; Robinson and Rapoport, 1987;Zucker et al., 1994Zucker et al., , 1995. Competitive binding studies with other amphiphi...

Resonance Raman Spectroscopic Study on Chiral Aggregation of Bilirubin-Bovine Serum Albumin Complex Formed at Liquid/Liquid Interface

“…At this time the molecular structure of BR was altered, as shown by changes in not only the dihedral angle and orbit overlap, 3,4 but also hydrogen bonds, as shown in Fig earlier stage, the induced CD spectra showed no obvious signals more than noises, which might suggest that the complexation interconverting (P+) to (M-) enantiomer is so slow at the interface that the chiral equilibrium between the (M+) and (P-) conformations was not shifted at this stage, though chloroform can be bound to BSA and may alter the complexation rate between BR and BSA at the interface. 7,8 With the increase in the reaction time, positive and negative Cotton effects became remarkable in the CD spectra. When the reaction time was near 40 min, the intensities of CD signals were almost invariable, indicating that the binding of BR to BSA and the aggregation of the BR-BSA complex were approaching their equilibria.…”

“…15,16 According to the exciton chirality rule, 15,16 dipyrrinone-dipyrrinone intramolecular exciton interactions (exciton coupling) coming from either of the BR conformation of (P+) and (M-) can generate CD spectra with opposite signs. It is possible that the equilibrium racemic state is broken and an induced optical activity can be generated, [3][4][5][6][7][8] because either enantiomer of the (P+) and (M-) forms of BR binds with an external chiral protein, such as BSA.…”

“…If the body does not produce an adequate amount of albumin, BR is sequestered directly under the skin, and even provoke BR encephalopathy by passing the blood-brain barrier and penetrating the phospholipid membrane of neurons. Although BR and its complex with serum albumin (SA) have been extensively investigated in bulk phases, [2][3][4][5][6][7][8][9][10][11] the complexation of BR with SA has never been studied at a liquid/liquid interface, which is recognized as being a model of a biological membrane, 12 and as a specific reaction field in the solvent extraction of metal ions. 13 In the present work, resonance Raman spectroscopy was applied to measure the microenvironment of an interfacial complex of BR with bovine serum albumin (BSA) and the aggregate of the complex at the organic/water interface.…”

Epigenetic DNA-methylation regulation of genes coding for lipid raft-associated components: A role for raft proteins in cell transformation and cancer progression (Review)

“…Removal of cholesterol leads to dissociation of most proteins from rafts and renders them non-functional (3)(4)(5)(6)(7)(8)(9)(10). The existence of rafts was first inferred from the differential trafficking of lipids and lipid-anchored proteins (PLAP-alkaline phosphatase, and HGinfluenza virus hemagglutinin) to the apical macro-domain of polarized epithelial cells (3,5,20), and later experimentally identified by determining with membrane-protein insolubility in cold non-ionic detergent, Triton X-100 (15,(20)(21)(22)(23). Thus, detection of complex formation with detergent resistant membranes (DRMs) became a useful approach to test whether or not a protein associates with rafts.…”

Effect of chloroform on complexation and chiral aggregation of bilirubin–bovine serum albumin at heptane/water interface

“…Therefore, the study of reactivity and physiological functions of BR has extensively been investigated. The study of the interaction of BR with serum albumin has been conducted in bulk phases [6][7][8][9][10][11][12], and on solid surfaces [13,14], but very few at liquid/liquid interface by far. Because the liquid/liquid interface can be regarded as a model of biological membrane [15] and a specific reaction field relevant to many complex chemical processes [16], we have investigated the adsorption and reaction of BR at liquid/liquid interface, and obtained some interesting results [17,18] that the interfacial complex of BR with bovine serum albumin (BSA) formed further the aggregates at the interface.…”