Protein ultracentrifugation

Fig. 5a - c. Molecular properties of bovine serum albumin as a function of the molar concentration, C3, of the denaturant guanidinium chloride (from [83D1]). a apparent isopotential specific volume (in cm g ) b extinction coefficient c intrinsic viscosity (in cm g ). For experimental details see the original reference. Reprinted from International Journal of Biological Macromolecules, Vol. 5, H. Durchschlag and R. Jaenicke, Partial specific volume changes of proteins ultracentrifugal and viscometric studies, pp. 143-148, 1983, with permission from Elsevier Science. 

Apart from tliese mainstream metliods enabling one to gain a comprehensive and detailed stmctural picture of proteins, which may or may not be in tlieir native state, tliere is a wide variety of otlier metliods capable of yielding detailed infonnation on one particular stmctural aspect, or comprehensive but lower resolution infonnation while keeping tlie protein in its native environment. One of tlie earliest of such metliods, which has recently undergone a notable renaissance, is analytical ultracentrifugation [24], which can yield infonnation on molecular mass and hence subunit composition and their association/dissociation equilibria (via sedimentation equilibrium experiments), and on molecular shape (via sedimentation velocity experiments), albeit only at solution concentrations of at least a few tentlis of a gram per litre. 

Fig. 2. Ultracentrifugal pattern for the water-extractable proteins of defatted soybean meal in pH 7.6, 0.5 ionic strength buffer. Numbers above peaks are approximate sedimentation coefficients in Svedberg units, S. Molecular weight ranges for the fractions are 2S, 8,000—50,000 7S, 100,000—180,000 IIS, 300,000—350,000 and 15S, 600,000—700,000 (9). The 15S fraction is a dimer of the IIS protein (10).

Separation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH-dependent. 

Techniques for study of higher orders of protein stmcture include x-ray crystallography, NMR spectroscopy, analytical ultracentrifugation, gel filtration, and gel electrophoresis. 

In addition to the determination of molar mass distributions and various molar mass averages there are many experiments, requiring sometimes sophisticated data evaluation, that can be carried out with an analytical ultracentrifuge. Examples are the analysis of association, the analysis of heterogeneity, the observation of chemical reactions, and protein characterization, to mention only a few. A detailed discussion is beyond the scope of this article, but there is excellent literature available [77-79,81,87-89]... 

B. Schmidt and D. Riesner, A fluorescence detection system for the analytical ultracentrifuge and its application to proteins, nucleic acids, viroids and viruses (in Ref. [77]). 

A) Fractured convex half and (B) concave half of Mab (PhytVSQDG (9 1 mol/ mol) vesicle containing BR (C) convex half and (D) concave half of protein-free vesicle. Bar = 50 nm. Samples were prepared at the initial concentration of 2 mM Mab (Phyt)2, 0.22 mM SQDG and 50pg/ml BR and concentrated by ultracentrifugation in 75 mM K2SO4 buffer solution. 

Lp(a), moving, like very low-density lipoproteins (VLDL), in the pre-(3,-lipoprotein fraction upon electrophoresis (B8, B9), but not floating like VLDL in ultracentrifugation (H29), partly resembles low-density lipoprotein (LDL). Its protein moiety consists of one glycoprotein molecule, named apolipoprotein (a)... 

Early ultracentrifuges were extremely expensive and were therefore located in only a small number of laboratories. Nevertheless, rapid strides were made in the theory and practice of ultracentrifugation. By 1940 the behavior of proteins with molecular weights ranging from that of cytochrome c (13.4 kDa) to serum globulin (ca.170 kDa) had been studied. 

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