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

Abstract

Resonance Raman spectroscopy assisted by centrifugal liquid membrane/circular dichroism (CLM-CD) and UV/Vis absorption spectroscopies was applied to measure the binding state of bilirubin (BR) in the complex with bovine serum albumin (BSA) formed at a heptane/water interface. The bisignate Cotton effects in the interfacial CD spectra and the red shift and linewidth increase of the BR absorption band around 450 nm indicated the formation of the BR-BSA complex at the interface and the chiral conversion of BR molecules in the aggregates. The resonance Raman spectra of BR observed at the interface suggested that the interfacial BR-BSA complex formed during the initial 15 min after the contact of the two phases had a similar structure with that in solution, but after 15 min were forming aggregates coexisting with solid micro-particles. These experimental results strongly suggested that the chiral interconversion of BR from (P+) conformation to (M-) conformation in the interfacial complex was accompanied by aggregation of the BR-BSA complexes. In the present study, resonance Raman microscopic spectrometry was proved to be highly useful for characterizing the solid like aggregate formed at the liquid/liquid interface. 3 The dashed lines show intramolecular hydrogen bondings that link the dipyrrinones to opposing propionic acids. θ denotes the dihedral angle (see the text for details).

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

“…The latter is presumably essential for the change in CD chirality of BR bound to BSA. The conformational change of BR can only be confirmed by the CD appearance and the red shift of the 450 nm band [17]. No inversion of CD sign of the interfacial BR-BSA complex at the initial reaction stage indicates that the BSA molecule of the inter- facial BR-BSA complex is located at the aqueous phase side of the interface and less affected by the CHCl 3 dissolved in the heptane phase.…”

“…After the attainment of the absorbance maximum (4 min 40 s), the absorbance gradually decreased, as well as the fluorescence intensity, which is discussed in the next section, and CD gradually appeared (2nd and 3rd process in Scheme 1), suggesting that the aggregates of BR-BSA complex formed slowly. After undergoing a flat plateau for CD amplitude or absorbance intensity (2nd and 3rd process in Scheme 1), the completion of the complex formation and subsequent aggregation of the complex and microparticles [17] (3rd process in Scheme 1) were suggested. As comparison, the time dependences of CD and absorbance at Cv = 16.7% were shown in Fig.…”

“…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. These findings are very important for understanding the interaction mechanisms of BR with BSA at biological membranes.…”

Structural Evaluation of the Aggregate of Palladium(II)-Pyridylazophenol Complex at the Heptane-Water Interface by the Centrifugal Liquid Membrane Method

“…which can be combined with other spectroscopic methods such as fluorometry [9], resonance Raman spectrometry [10][11][12][13], and circular dichroism spectropolarimetry [14,15]. Several studies on the interfacial aggregate formation of metal complexes have been carried out using CLM [16][17][18][19][20][21].…”

Microscopic Measurement of Circular Dichroism Spectra

“…1,2 Usually, isotropic and homogeneous solution samples are employed for measuring the CD spectra; however, in some cases CD spectra of samples with unknown inhomogeneity should be measured. For example, we have observed that some chiral aggregates grown at liquid/liquid interfaces show chiral activity, [3][4][5][6][7][8] but the chirality of the individual micro-aggregate can not measured by a conventional CD spectropolarimeter. Also, almost all biological samples have inhomogeneity, because they consist of various tissues.…”