Investigation of the thermally structural denaturation of bovine serum albumin by a home-made optical heterodyne polarimeter

Abstract

ABSTRACT Bovine serum albumin (BSA) solutions (2.5 %, PH 7.2) under heat treatment can result in st ructural changes. Some of the Þ -helices are transformed at 67  to random coils resulting in an increased rotation angle. With subsequent heating, the transformed random coils may once again transform to non-native s -sheets and restore the optical rotation angle. These two states are all reversible. However, when the heating temperature goes up to 69  the denatured BSA starts to transform into a rigid network and becomes an irreversible state. The reversible and irreversible temperatures of BSA were determined and discussed. Keywords: Heterodyne polarimeter, bovine serum albumin, thermal denaturation. 1. INTRODUCTION Bovine serum albumin, a highly complex protein, is one of the most frequently studied proteins in food systems and in immunology analysis [1-5]. Previous studies indicate that the secondary structures of BSA contain about 50-55% .-helix, 15-18% -sheet and 27-35% random coils [6]. In contrast , based on X-ray crystallography, there are no -sheets in the structure of the native form [7]. When heat is applied to BSA, the kinetic energy and the molecular vibration increase, and as a result, the hydrogen bonds and non-polar hydrophobic interactions are in terrupted [8-9]. Harmsen and Braam [10], using infra-red and optical rotation dispersion (ORD) spectra concluded that alkali or heat denaturation caused a partial loss of alpha-helical structure with no formation of -sheets. However, from their infra-red spectra, a shoulder appeared for BSA heated above 72°C at 1620 cm-1, which is indicative of beta-sheet formation. Clark et al. [11] using infra-red and Raman spectra reported that gels formed from heat and that chemically denatured proteins arose from the interaction of regions of beta-sheets. They also noted that a shoulder was visible on their infra-red spectra at 1620 cm-1 for BSA heated to 75°C and above. Further studies have indicated that BSA when heat-treated, goes through two structural stages. The first stage is reversible while the second stage is irreversible, but it does not necessarily result in a complete destruction of the ordered structure [12-13]. Results from circular dichroism (CD) and infra-red (IR) spectroscopy indicated that -sheets were formed when albumin was heated above 65°C [14], 70°C [13]. Recently, Teepakorn et al. [15] using optical rotation (OR) and transmitted light (TL) methods reported that the denaturation temperatures (Td) obtained from OR measurements were consistent with reported differential scanning calorimetry (DSC) values. In the present study, instead of measuring the sample’s spectra by IR [10-14], CD [13], DSC [15], as mentioned above, or other methods such as X-ray diffraction crystallography [ 16-17], and optical rotation disp ersion [18-19], we proposed a novel method to measure the changes in the optical activity of the sample. We used a home-made optical heterodyne polarimeter, which could enhance the optical rotation angle, to determine the temperature required to start the conformational change of the BSA as well as the reversible and the irreversible stages.