Protein volume, determination

Lactoglobulin A in 40% 2-Chloroethanol. Previous light scattering and differential refractometry measurements (8, 23) have shown that / -lactoglobulin exhibits strong preferential interactions with solvent components in the water-2-chloroethanol system. Since the preferential interaction between protein and 2-chloroethanol in this system was found to be maximal at 40% (v/v), the effect of this interaction on the partial specific volume of the protein was determined. 

The product of the crystal density and the unit-cell volume (determined from crystallographic analysis, Chapter 4) gives the total mass within the unit cell. This quantity, expressed in daltons, is the sum of all atomic masses in one unit cell. If the protein molecular mass and the number of protein molecules per unit cell are known, then the remainder of the cell can be assumed to be water, thus establishing the proteinlwater ratio. 

Isolation of Labelled Glycolipids. Cells were sonicated in a small volume of saline and the total protein was determined on an aliquot by the method of Lowry et. al. (5). Total lipids were extracted with 20 volumes of chloroform methanol (2 1) filtered, and the residue re-extracted with 10 volumes of chloroform methanol water (1 2 0.15). Extracts were combined and concen-... 

Lovenberg, Buchanan, and Rabinowitz 65) showed that the molecular weight of C. pasteurianum ferredoxin is about 6000, based on sedimentation velocity and sedimentation equilibrium ultracentrifugation determinations and on amino acid analysis. The sedimentation coefficient, S2o,w was 1.4, and the partial specific volume, determined according to the method of Hvidt et al. 59) was 0.63, as compared to the value of 0.71 observed for most proteins. Similar investigations showed that ferredoxins from four other clostridia Lovenberg, Buchanan, and Rabinowitz 65)) and from a photosynthetic bacterium (Bachofen and Arnon 12)) also had a molecular weight of about 6000. 

However, it is possible to quantify the AFM images (Mackie et al., 1999). Areas occupied by either protein or surfactant can be calculated and, when combined with measurements of the thickness of the protein network, the protein volume can be determined. Figure 15.3 reveals the true nature of the displacement process. The protein volume remains constant until the network snaps, indicating that no protein is lost from the interface into the bulk until the protein network has been broken. 

The volume opens with a report by L.D. Field on Multiple Quantum NMR of Partially Aligned Molecules following this is an account on Solid-State NMR Studies of Molecular Motion by M.J. Duler C. Odin reviews NMR Studies of Phase Transitions Application of Multi-way Analysis to 2D NMR Data is covered by H.T. Pedersen, M. Dyrby, S.B. Engelsen and R. Bro the final contribution is on High Resolution Protein Structure Determination by NMR and it is provided by H. Takashima. My sincere thanks go to all of these reporters and to the production staff at Elsevier for their assistance in the creation of this volume. 

The molecular weight of the protein is determined by comparing its relative elution volume (Ve - Vc/Vg) to those of several standard molecules (Figure 5D). Ve is the solvent volume required for the elution of a solvent from the column. 

Edman, Pehr Victor (1916-1977), a pioneer in the field of peptide and protein chemistry. Already for his Ph.D he had isolated and purified angiotensin. In 1947, he accepted an associate professorship at the University of Lund (Sweden), and in 1957 Edman came to Melbourne as the first John Holt director of research at St. Vincent s School of Medical Research. In 1972, he moved to Germany, having been appointed Professor at the Max-Planck-Institute for Biochemistry at Martinsried. He is the inventor of an important method for protein structure determination, named the Edman degradation [F. J. Morgan, Edman, Pehr Victor (1916-1977), Austr. Diet. Biograph. 1996, Volume 14, pp. 78-79]. 

Analysis typically involves three steps. First, exoproteins are isolated from bacterial cultures. The aim of this process is to retain as many proteins as possible while eliminating components that inhibit isoelectric focusing. To do so, proteins are enzymatically treated and precipitated to remove salts and other non-protein components. Second, the proteins are resolved by one- or two-dimensional gel electrophoresis (see Fig. 1). Third, proteins are visualized and the composition and quantities of protein spots determined. The protocol described below uses a mini-gel format that is relatively fast and requires small culture volumes compared with large-format two-dimensional gel electrophoresis. 

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