Thermal effect of sonication on bovine serum albumin

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Figure A2 Variations of inner side-chain length (R2-R1) with radius of curvature. In the case of Out (C) the full line represents the calculated length of the 6-methyl-hept-2-yl residue and should be compared with the lower broken line representing the estimated maximum extension of this side chain. The other full lines represent the calculated distances occupied by mixed fatty acid ester and C- 17 side chains (Anti) (B) or fatty acid ester chains (In) (A) which can exceed the upper broken line representing the fully extended length of linoleate

(hydrated); P3, 0.37; P2, 0-305;pl, 0.295; P0, 0.3346, for water density. As the shell radii of the cholesteryl ester layers are decreased the inner side of each layer becomes progressively compressed resulting in a forced extension of the hydrocarbon section towards the centre of the structure. A simple criterion of acceptability is that the radial distance required to hold the mass present must not exceed the fully extended length of the C-17 or fatty acid ester chain. Figure A2 shows that limit for the three layer types. Using the R4 values of a 105 ~ sphere (52, 56 and 46 •& for Anti, Out and In outer layers, respectively), it can be seen that the choice of inner layer conformation is restricted. In conformation is not permissible at the inner layer, Anti may be acceptable only as Out-Anti, and Out inner layers are acceptable with Out or Anti outer layers. Normalized scattered intensities of disoriented dispersions of the model L D L were calculated by means of the appropriate equation of ellipsoidal scattering 2° as modified for the nested scheme indicated:

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irrelevant if all hydrocarbon chains have equal density. The small amount of matter ( 4 1% of L D L mass) present in the centre of discrete models makes an insignificant contribution to X-ray scattering. It was assumed to have the electron density of triglyceride. Shell radii were calculated at various values of the parameters p, P3 and Y. The proportion of triglyceride is low and calculations made for Y = 0 (all the triglyceride confined to the hatched centre of Figure A1) or Y=0.06 were only marginally different. Density values were derived by testing possible values in diffraction calculations. Suitable values are:ps, 0.45 (unhydrated) or 0.42

Thermal effect of sonication on bovine serum albumin Koichiro Aoki, Shigenori Maezawa, Tooru Ito and Koichi Hiramatsu Department of Synthetic Chemistry, Gifu University, Kagamigahara, Gifu Pref., 504, Japan (Received 5 March 1980; revised 30 April 1980)

The effect of ultrasonic irradiation on bovine serum albumin (BSA) has been studied by several workers I -T However, the results obtained are not consistent because of differences in the irradiation conditions. In this study, BSA solutions irradiated at pH 8.3 were analysed by disc gel electrophoresis. The temperature rise in the solution due to irradiation caused aggregation of the BSA molecule. This is one of the effects on BSA of ultrasonic irradiation, and may be interesting since sonication is used 0141-8130/81/0201434)2502.00 ©1981 IPC Business Press

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+(P2 - Pl)[ R1]3f~'(R1) in which ~'(Rb)= ¢~{2n[Rb3s[l + (v2 - 1)x2]} 1/2 where is the function for the structure amplitude of a plain sphere. The averaged scattered intensity is: 1

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frequently in the extraction of membrane proteins. Armour crystallized BSA (lot no. M72603) and 0.1 M Tris-HCl buffer, pH 8.3, were used. A freshly prepared 0.5~ BSA solution was irradiated under atmospheric pressure. The ultrasonic wave was generated by a USV150V generator from Ultrasonic-wave Ind. Co. As.shown in Figure 1, 5 ml of BSA solution was placed directly onto the oscillator and the assembly immersed in a water bath at 25°C. The oscillator was made from barit~m titanate and its surface was coated with epoxy resin paint; its diameter was 6 cm. Standard disc gel.electrophoresis was performed following the procedure of Ornstein s and Davis 9. After electrophoresis, the gel was stained by Amido black 10B and then destained. Figure 2 shows the results from disc gel electrophoresis. For native BSA (A in Figure 2) there are two zones, labelled 1 and 2'. The component in zone 1 (component 1) is native monomer, while that in zone 2' is a dimer, an

Int. J. Biol. Macromol., 1981, Vol. 3, April

143

Notes to the Editor impurity found in commercial BSA. The relative mobility of component 2' is 0.59-0.61, if the mobility of component 1 is taken to be 1.00. The pattern of BSA heat-denatured at 65°C and pH 9.1 1o (B in Figure 2) shows several zones which migrate more slowly than native BSA. Components 1' and 1" are modified monomeric albumins, and component 2 is a dimer of BSA. Components 3 and 4 are probably trimer and tetramer, respectively. The relative mobilities of each component are indicated to the right of each zone. Components 2' and 2 are different dimers: the latter is formed by disulphide bonding, and the former is formed by some unknown bond 1~. The mobility of dimer 2' is ~0.60 and that of dimer 2 is ~0.50. Thus dimer 2 moves more slowly than dimer 2'. The pattern of BSA irradiated at 1.6 MHz and 50 W for 30 min (C in Figure 2) shows several zones which move more slowly than component 1. Similar patterns were obtained when BSA solution was irradiated under different conditions, i.e. in the range 0.2-1.6 MHz and 50150 W. Relative mobilities are given for each zone. When C and B in Figure 2 are compared it is found that components 1", 1', 2, 3 and 4 were formed during sonication, and that additional components having mobilities of 0.59 and 0.45 were also formed. This implies that the temperature rise in BSA solution after sonication caused aggregation of the BSA molecule. Indeed, a thermistor placed in the BSA solution indicated that the temperature rose to 40-50°C. When running water or icecold water was circulated around the assembly in Figure 1 during sonication, the temperature rise was prevented. Under these conditions, aggregates were not formed. If the present results are taken into consideration, the results in the literature can be explained more reasonably. In the study by Hess et al. s, BSA in 0.1 M KC1 was sonicated at 9 kHz and 50 W for 120 rain. A variety of procedures have been used to examine sound-treated

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solutions of BSA. Among them, values of molecular weight of BSA determined by light scattering were markedly different for treated and untreated BSA. The most plausible explanation for the effects of highfrequency sound appeared to be aggregation. Searcy et al. 6 sonicated human serum albumin in saline solutions at 20 kHz for 10 min. They analysed the treated solution in a Tiselius electrophoresis apparatus and found that such treatment of the albumin solution caused ~ 16% of the protein to migrate less rapidly anodally. Both of these results can be interpreted more exactly by the patterns shown in Figure 2,

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Chambers, L. A. and Flosdorf, E. W. J. Biol. Chem. 1936, 114, 75 Prudhomme, R. O. and Graber, O. Bull. Soc. Chim. Biol. 1947, 29, 122 Kaning, K. and Kunkel, H. Z. Physiol. Chem. 1958, 309, 162 Kaning, K. Z. Physiol. Chem. 1958, 309, 171 Hess, E. L., Chun, P. W. L. and Crowley, R. L. Science 1964, 143, 1176 Searcy, R. L. and Hines, L. R. Experientia 1969, 25, 914 El'piner, 1. E. in 'Ultrasound. Its Physical, Chemical and Biological Effects', Consultant Bureau, New York, 1964, pp. 170180 Ornstein, L. Ann. N.Y. Acad. ScL 1964, 121, 321 Davis, B. J. Ann. N.Y. Acad. Sci. 1964, 121, 404 Aoki, K., Sato, K., Nagaoka, S., Kamada, M. and Hiramatsu, K. Biochim. Biophys. Acta 1973, 328, 323 Janatova, J., Fuller, J. K. and Hunter, M. J. J. Biol. Chem. 1968, 243, 3612