Proteins intrinsic viscosity

The ionic strength dependence of intrinsic viscosity is function of molecular structure and protein folding, ft is well known that the conformational and rheological properties of charged biopolymer solutions are dependent not only upon electrostatic interactions between macromolecules but also upon interactions between biopolymer chains and mobile ions. Due electrostatic interactions the specific viscosity of extremely dilute solutions seems to increase infinitely with decreasing ionic concentration. Variations of the intrinsic viscosity of a charged polyampholite with ionic strength have problems of characterization. 

Diffusion and sedimentation measurements on dilute solutions of flexible chain molecules could be used to determine the molecular extension or the expansion factor a. However, the same information may be obtained with greater precision and with far less labor from viscosity measurements alone. For anisometric particles such as are common among proteins, on the other hand, sedimentation velocity measurements used in conjunction with the intrinsic viscosity may yield important information on the effective particle size and shape. ... 

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

The measurements of chain stiffness of denatured proteins are made in the presence of a strong denaturant, such as 8 M urea or 6 M GdmCl, in which peptide H-bonds are weak and peptide helices unfold (Scholtz et al., 1995 Smith and Scholtz, 1996), and the possible presence of (/-helices or /3-hairpins is not an issue in these denaturants. The careful and thorough measurements of intrinsic viscosities made by Tanford and co-workers (1968), discussed above, yield a substantially lower estimate for chain stiffness than the work of Flory and co-workers. A comparison is made by Tanford (1968) between the proportionality coefficient... 

Finally, for biological molecules that are macromolecules, such as most proteins, Eq. (4.23) can also be used to relate the relative viscosity to the intrinsic viscosity of the solution and the macromolecule concentration ... 

F ure 4.20 Variation of intrinsic viscosity of aqueous protein solutions with axial ratio and extent of solvation. Reprinted, by permission, from P. Hiemenz, Polymer Chemistry, p. 598. Copyright 1984 by Marcel Dekker, Inc. 

The intrinsic viscosity of native and denatured soy proteins have been measured (13). These values, which reflect the hydrodynamic properties oT the protein molecules at infinite dilution, are of little interest to us as far as functionality is concerned. What is of interest is the apparent viscosity of concentrated slurries. In these slurries, the intermolecular protein-protein interactions dominate and are primarily responsible for the observed viscosity behavior. 

EXAMPLE 4.4 Extent of Hydration of a Protein Molecule from Intrinsic Viscosity Measurements. Suppose an aqueous solution of a spherical protein molecule shows an intrinsic viscosity of 3.36 cm3 g 1. Taking p2 = 1.34 g cm 3 for the dry protein, estimate the extent of hydration of the protein. 

Solution It is apparent from the units of b] that solute concentration has been expressed in g/cm3. Dividing this concentration by the density of the unsolvated protein converts the concentration to dry volume fraction units. Since the concentration appears as a reciprocal in the definition of [17], we must multiply bl by p2 to obtain (lAA Mb/r/o) - 1]. For this protein the latter is given by (3.36)(1.34) = 4.50. If the particles were unsolvated, this quantity would equal 2.5 since the molecules are stated to be spherical. Hence the ratio 4.50/2.50 = 1.80 gives the volume expansion factor, which equals [1 + (mhb/m2)(p2/p )]. Therefore (m, tblm2) = 0.80(1.00/ 1.34) = 0.6O. The intrinsic viscosity reveals the solvation of these particles to be 0.60 g HaO per gram of protein. ... 

Finally, we note that both solvation and ellipticity can occur together. The contours shown in Figure 4.13a illustrate how various combinations of solvation and ellipticity are compatible with an experimental intrinsic viscosity. The particle considered in Example 4.4 has an intrinsic viscosity of 4.50 and was calculated to be hydrated to the extent of 0.60 g HzO per gram of protein. The same value of [17] is also compatible with nonsolvated ellipsoids of... 

FIG. 4.13 Intrinsic viscosity of a protein solution (a) variation of the intrinsic viscosity of aqueous protein solutions with axial ratio a/b and extent of hydration mlb/m2 (redrawn from L. Oncley, Ann. NY Acad. Sci., 41, 121 (1941)) (b) superposition of the [r ] = 8.0 contour from Fig. 4.13a and the f/f0 = 1.45 contour from Figure 2.9. The crossover unambiguously characterizes particles with respect to hydration and axial ratio. 

The ash content, protein concentration, maximum intrinsic viscosity, equivalent weight and rotatory capacity are some of the features specific to each species of Acacia exudate. 

Nuclease behaves like a typical globular protein in aqueous solution when examined by classic hydrodynamic methods (40) or by measurements of rotational relaxation times for the dimethylaminonaphth-alene sulfonyl derivative (48)- Its intrinsic viscosity, approximately 0.025 dl/g is also consistent with such a conformation. Measurements of its optical rotatory properties, either by estimation of the Moffitt parameter b , or the mean residue rotation at 233 nin, indicate that approximately 15-18% of the polypeptide backbone is in the -helical conformation (47, 48). A similar value is calculated from circular dichroism measurements (48). These estimations agree very closely with the amount of helix actually observed in the electron density map of nuclease, which is discussed in Chapter 7 by Cotton and Hazen, this volume, and Arnone et al. (49). One can state with some assurance, therefore, that the structure of the average molecule of nuclease in neutral, aqueous solution is at least grossly similar to that in the crystalline state. As will be discussed below, this similarity extends to the unique sensitivity to tryptic digestion of a region of the sequence in the presence of ligands (47, 48), which can easily be seen in the solid state as a rather anomalous protrusion from the body of the molecule (19, 49). 

Tanford, C., Kawahara, K. and Lapanje, S. (1967). Proteins as random coils. I. Intrinsic viscosities and sedimentation coefficients in concentrated guanidine hydrochloride. Journal of the American Chemical Society, 89, 729-736. 

Based on properties in solution such as intrinsic viscosity and sedimentation and diffusion rates, conclusions can be drawn concerning the polymer configuration. Like most of the synthetic polymers, such as polystyrene, cellulose in solution belongs to a group of linear, randomly coiling polymers. This means that the molecules have no preferred structure in solution in contrast to amylose and some protein molecules which can adopt helical conformations. Cellulose differs distinctly from synthetic polymers and from lignin in some of its polymer properties. Typical of its solutions are the comparatively high viscosities and low sedimentation and diffusion coefficients (Tables 3-2 and 3-3). 

The formation of polymer-polymer complexes as a rule is observed in aqueous media5,33. The viscosity of complexes in water is about 0.05-0.10 dl/g and close to that of globular proteins. Aqueous solutions have some features low intrinsic viscosity values are independent of the matrix molecular weight, the absence both of the concentration dependence of the reduced viscosity and the polyelectrolyte anomaly, and high values (about 30 s) of the sedimentation constant. 

The stability of complexes formed in non-aqueous media against the destructive action of organic solvents has been studied (DMSO). The PAA-PVP polycomplex retains its compact structure up to 40 vol-% content of DMSO in the mixture with methanol (Fig. 16, curve 2) this follows from the low values of the intrinsic viscosity. A further increase of the DMSO content results in a sharp rise of the viscosity i.e. the complex dissociates. That the complex breaks down in pure DMSO is confirmed by the following facts the intrinsic viscosity of the PAA-PVP system is an additive function of the viscosity of individual polymers in this solvent while in methanol [ 7] of the PAA-PVP system is close to the viscosity of globular proteins. It is of interest that... 

In aciueous solutions containing high concentrations of LiBr or CaCb, the specific rotation of poly-E-proline II approaches [a] = —240°. Under these CH)nditions the intrinsic viscosity of the highly a.symmetric form II structure decreases markedly to values in the range of the globular proteins and it can be assumed that the configurational contribution to rotation of form II has been eliminated. 

Intrinsic Viscosities of Several Proteins and Polymers in Solutions ... 

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