Abstract Bovine serum albumin (BSA) under ultrasonic irradiation was carried out using circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR). Quantitative analysis of the FT-IR absorbance spectra at amide I (1600-1700 cm^-1) as well as far UV circular dichroism data showed a slight increment in helicity along with a slight decrease in the a % helix. Circular dichroism spectroscopy and FT-IR analysis indicated changing in the secondary structure of BSA.
Keywords BSA; ultrasonic; CD spectra; FT-IR spectra

1 Introduction
Ultrasonic frequencies can be divided into low-frequency (2 × l0⁴- 2 × l0⁵ Hz), intermediate frequency (10⁵ - l0⁷ Hz) and high-frequency (10⁷ - l0⁹ Hz) ^[1]. Low-frequency ultrasound has been observed to be more efficacious than high-frequency ultrasound in the physical aspects of ultrasound ^[2,3]. Therapeutic ultrasound at lower frequencies has also been used experimentally for biological macromolecules with promising results. Serum albumins are one of the most abundant proteins in blood plasma, which are the major soluble protein constituents of the circulatory system. They play an important role in the transport and deposition of many drugs molecules in the blood^[4]. Bovine serum albumin (BSA) is a single-chain 582 amino acid globular non-glycoprotein cross-linked with 17 cystine residues (eight disulfide bonds and one free thiol) ^[5,6]. It is a typical kind of large globular protein and the secondary structure is mainly composed of a-helicity. In our previous work, BSA is selected as a model protein molecule because of its lone-standing interest in protein community^[7]. Consequently, it is important to study on the conformation of bovine serum albumin under irradiation of low frequency ultrasound ^[8]. In this paper, changes in the secondary structure of BSA has been extensively studied via circular dichroism (CD) and Fourier transform infrared spectroscopy( FT-IR ).

2 Materials and methods
2.1 Materials
BSA was purchased from Sigmia Company and used without further purification and its molecular weight was 66210. NaCl solution (0.1 mol·L^-1) was used to maintain the ionic strength in 0.1 mol·L^-1 PBS buffer (pH = 7.43). BSA (1.0 × 10^-6 mol·L^-1) solution was prepared in pH = 7.43 PBS buffer solution and kept in the dark at 4ºC. The pH was checked with Sartorius PP-20 standardized pH meter (Germany). All starting materials were of analytical reagent grade and doubly deionized distilled water was used throughout. Ultrasound was applied by using a SW-1006 low frequency ultrasound apparatus.
2.2 FT-IR spectroscopy
FI-IR measurements were carried out at room temperature on Perkin-Elmer FT-IR spectrometer (America) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detectorand a KBr beam splitter. All spectra were taken via the attenuated total reflection (ATR) method with resolution of 4 cm^-1 and 60 scans. Spectra processing procedures: spectra of buffer solution were collected under the same conditions. Then, the absorbance of buffer solution from the spectra of sample solution was subtracted to get the FT-IR spectra of proteins. The subtraction criterion was that the original spectrum of protein solution between 2200 and 1800 cm^-1 was featureless^[9].
2.3 Circular dichroism (CD) spectropolarimetry
All CD measurements were obtained employing Jasco-810 circular dichroism ( Japan, Jasco company ). The far UV region was scanned between 190 and 250 nm.

3 Results and discussion
3.1 FT-IR spectroscopy

Fig.1 FT-IR spectra and difference spectra of BSA (a) and BSA after ultrasonic irradiation 20 min(b); 40 min (c) in buffer solution in the region of 1800–1300 cm^-1, [BSA] = 1.0 × 10^-3 mol·L^-1. T = 298 K, pH = 7.43.

The FT-IR spectra of the BSA and BSA after ultrasonic irradiation are shown in Fig. 1. The spectrum in Fig. 1.a, b, and c was obtained by subtracting the absorption of the PBS buffer from the spectrum of BSA solution, BSA solution after frequency ultrasound irradiated 20 and 40 min, respectively.
    The protein amide I in the region 1600-1700 cm^-1 (mainly C-O stretch) and amide II band ≈1548 cm^-1 (C–N stretch coupled with N–H bending mode) both have a relationship with the secondary structure of protein, and amide I band was more sensitive to the change of protein secondary structure than amide II ^[10,11]. As shown in Fig. 1, after ultrasonic irradiation, the amide I band of BSA moved from 1638.0 to 1644.6, 1648.0 cm^-1 and the amide II band moved from 1559.9 to 1550.3, 1544.7 cm^-1, which indicate that the protein secondary structure has been changed under ultrasonic irradiation.
   A quantitative analysis of the protein secondary structure for the free BSA and low frequency ultrasound irradiated BSA is given in Fig. 2. and table 1. According to the literature ^[12,13], before estimation of percentage content of each secondary structure, the component bands should be assigned. The bands range 1610-1640 cm^-1 are generally assigned to b-sheet, 1640-1650 cm^-1 to random coil, 1650-1658 cm^-1 to a-helix and 1660-1700 cm^-1 to b-turn structure. The percentages of each secondary structure of BSA can be calculated based on the integrated areas of the component bands in amide I.



Fig. 2. Decomposition of the infrared spectra of 1.0 × 10^-3 mol·L^-1 BSA at 50mmol·L^-1 phosphate buffer (pH = 7.43) in the absence (a) and after ultrasonic irradiation 20 min (b); 40 min (c).

    As shown in table 1, BSA contained a-helix reduced from 55.78% to 45.15%, 42.40%，b-sheet from 35.81% to 36.75%, 38.03%, b-turn changed from 8.41% to 18.01%, 19.57%.

3.2 CD spectroscopy

Fig. 3 CD spectra of 1.0 × 10^-6 mol·L^-1 BSA in PBS(a) and BSA after ultrasonic irradiation 20 min(b); 40 min (c).

The CD spectra were also examined to ascertain the changes of BSA secondary structure under irradiation of low frequency ultrasound with spectrum in PBS buffer, the CD spectrum were presented in Fig.3 .
    The CD results were expressed in terms of the mean residue ellipticity (MRE) in degcm²dmol^-1 according to the following equation (1), (2) ^[14]
[q]_MRE208 = q_obs( mdeg)/(10 × n × l× Cp) (1)
    where Cp is the molar concentration of the protein, n is the number of amino acid residues (582) and l is the path length (0.1 cm).The a-helical content of BAS was calculated from the [q] value at 208 nm using the equation as described by Lu et al.^[15]
a% helix = [([q]_MRE208 － 4000)/(33000 － 4000)] × 100 (2)
    The calculating results exhibited a reduction of a-helix structures from 50.26% to 49.38%, 48.39% frequency ultrasound irradiated 20 and 40 min, respectively. The results of CD and FT-IR spectroscopy showed that the secondary structure of BSA was changed after irradiation of low frequency ultrasound.

低频超声波作用下牛血清白蛋白的构象研究
余静 张新波 边贺东 于青 梁宏 田建嬝
药用资源化学与药物分子工程教育部重点实验室，广西师范大学，541004，桂林
国家自然科学基金（20671023), 广西自然科学基金(0731052)，药用资源化学与药物分子工程教育部重点实验室资助项目。
摘要 应用傅立叶红外(FT-IR)和园二色光谱法(CD)研究了在生理pH 条件下牛血清白蛋白(BSA)在超声波作用下的构象变化。傅立叶红外光谱酰氨Ⅰ带（1600-1700cm^-1）和远紫外圆二色光谱数据分表明经低频超声波作用下BSA的a-螺旋结构的含量降低，其二级结构发生了改变。
关键词 牛血清白蛋白（BSA）；低频超声波；园二色光谱(CD)；傅立叶红外光谱(FT-IR)