Evaluation of nonideality from gel chromatographic partition coefficients

Damme, Markus; Murphy, William H.; Comper, Wayne D.; Preston, B. N.; Winzor, Donald J.

doi:10.1016/0301-4622(89)80014-6

Cited by 11 publications

(5 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…c) The value of 0.98 ( & 0.30) nm that is obtained for rf from the abscissa intercept (Fig. 4a) is certainly greater than the unhydrated rod radius of 0.3 nm that was assumed in earlier gel chromatographic studies [41,571, but agrees well with the values of 0.8 -1.3 nm inferred [59] from the effects of dextrans T80-T2000 on the sedimentation rates of xcrystallin. Even in the absence of dextran hydration, the representation of a flexible polymer as a rigid rod is bound to lead to an apparent radius greater than that inferred from the static configuration of a space-filling model of the polysaccharide chain.…”

Section: E-xcluded Volume Effects With Proteins and Linear Polymerssupporting

confidence: 85%

“…The partition coefficient, eA, obtained from Eqn (I), was then combined with the mass concentration of polymer, cp, to determine a second virial coefficient, PAPI via the expression which entails assumptions that self-interaction of the protein is contributing negligibly to the thermodynamic nonideality and that the polymer is excluded from the gel phase [41]. Due to adoption of the mass-concentration scale for expressing the space-filling effects of polymer, the second virial coefficient is defined in terms of volume/unit mass.…”

Section: Second Virial Coej$cients For Proteinldextran Interactionsmentioning

confidence: 99%

“…The following Stokes radii were used: cytochrome c, 1.65 nm [51]; cc-lactalbumin, 1.73 nm [52]; ribonuclease, 1.84 nm [47]; carbonic anhydrase, 2.30 nm [53]; pepsin, 2.74 nm [54]; horseradish peroxidase, 3.03 nm [55]; ovalbumin, 2.92 nm [46]: sorbitol, 0.37 nm [56]. The broken line in (a) is the relation presumed to apply in earlier investigations [41,571 has been assigned to dextran T70 [7]. Similar considerations of the second virial coefficient (A,) of 4.2 x mol cm3 g-' reported [50] for the present batch of dextran T2000 yield rp z 44 nm.…”

Section: E-xcluded Volume Effects With Proteins and Linear Polymersmentioning

confidence: 99%

“…An experimental test of this prediction is presented in Fig. 4a, which summarizes the results of covolume determinations of dextran T70 and a range of proteins by gel chromatography on Sephadex G-75: an earlier value for sorbitol [41] is also included. Several points should be noted.…”

Section: E-xcluded Volume Effects With Proteins and Linear Polymersmentioning

confidence: 99%

See 3 more Smart Citations

Thermodynamic nonideality in macromolecular solutions

Shearwin

Winzor

1990

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Second virial coefficients and hence covolumes for self-interaction of five proteins, viz. ribonuclease, ovalbumin, bovine serum albumin, catalase and a-crystallin, have been determined by analyzing the concentration dependence of the partition coefficient obtained from frontal chromatographic studies on either Fractogel TSK HW55 or porous glass beads. The resulting estimates of the effective radii essentially duplicate their Stokes counterparts and thereby provide further justification for assuming the approximate identity of the thermodynamic and hydrodynamic radii of hydrated globular proteins. Gel chromatographic evaluation of second virial coefficients for protein/dextran systems has led to elimination of the sphere/sphere model as a valid thermodynamic description of the space-filling effects in protein/polymer mixtures, since it does not predict the observed independence of covolume, expressed per unit mass of polymer, upon size of the polymer. This requirement is met by the sphere/ rod model [Edmond, E. & Ogston, A. G. (1968) Biochem. J. 109, 569-5761 and also by the sphere/flexiblesegment model [Hermans, J. (1982) J . Chenz. Phys. 77, 2193. Furthermore, similar studies of the effect of solute radius on covolume for interaction with dextran T70 attest to the adequacy of either model for predicting the thermodynamic nonideality arising from the inclusion of dextrans in protein solutions, and also provide the relevant calibration of the model.Studies of the effects of inert solutes on macromolecular interactions have led to the prediction and demonstration that self-association is enhanced by high concentrations of such solutes [l -61. In addition, advantage may be taken of the space-filling effects of inert solutes to detect enzyme isomerization [7], be it a substrate-induced [8-111 or a pre-existing [12 -141 phenomenon. Such thermodynamic nonideality of proteins/enzymes may be expressed quantitatively in terms of composition-dependent activity coefficients based on statistical-mechanical application of the excluded volume concept [15 -181. A quantity central to the prediction of thermodynamic nonideality is the covolume, i.e. the minimal volume in which two molecules can be located. In calculations of covolume, globular proteins have been regarded either as spheres or as ellipsoids of revolution [19, 201, whereas solutes such as poly(ethy1ene glycol) and dextran have been modelled as long rods [15] or as flexible segmented chains [17, 181. The major problem encountered in experimental studies of thermodynamic nonideality is thus not a lack of theoretical expressions for application but rather a lack of information on the magnitudes of parameters for substitution therein.In most studies of thermodynamic nonideality in protein solutions the stance has been taken that the Stokes radius of a globular protein should provide a reasonable estimate of its effective hydrated radius for use in covolume calculations [6 -12, 21, 221; however, this approximation is open to criticism on the grounds that a thermodynam...

show abstract

Section: E-xcluded Volume Effects With Proteins and Linear Polymerssupporting

confidence: 85%

Section: Second Virial Coej$cients For Proteinldextran Interactionsmentioning

confidence: 99%

Section: E-xcluded Volume Effects With Proteins and Linear Polymersmentioning

confidence: 99%

Section: E-xcluded Volume Effects With Proteins and Linear Polymersmentioning

confidence: 99%

See 2 more Smart Citations

Thermodynamic nonideality in macromolecular solutions

Shearwin

Winzor

1990

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…A gel chromatographic method has been used to characterize the nonideal interaction between a small polymer (PEG 4000) and each of two proteins (156). This method has two advantages over dialysis equilibrium, to which it is thermodynamically equivalent: measurements are much more rapid, and the technique may be utilized even when the two solutes are not completely separable, so long as they partition sufficiently distinctly on a given size exclusion gel matrix.…”

Section: Protein In Ti-iementioning

confidence: 99%

Macromolecular Crowding: Biochemical, Biophysical, and Physiological Consequences

Zimmerman¹,

Minton²

1993

Annu. Rev. Biophys. Biomol. Struct.

1,435

1,321

View full text Add to dashboard Cite

Measurements of biochemical rates and equilibria are conventionally carried out under relatively idealized conditions selected to minimize effects of nonspecific interactions between the constituents of the reaction unit (i.e. the reactants, products, and transition-state complexes) and other species that do not ~lirectly participate in the reaction under study. Concentration-dependent apparent rate or equilibrium constants are then extrapolated to the limit of infinite dilution in order to extract intrinsic quantities reflecting the properties of isolated reactant and product molecules in an idealized bath of solvent. Biological media differ from the idealized solvent bath in two important ways. First, a biological medium is likely to contain a high total concentration of nominally soluble macromolecules. Sometimes a single species at a high concentration predominates, such as hemoglobin within red cells (~350 g/liter) (see 3). More commonly, a medium may contain variety of macromolecular species, none of which taken individually may be present at high concentration, but which taken collectively occupy a substantial fraction of the total volume of the medium [e.g. ~ 340 g/liter

show abstract

Partitioning of flunitrazepam into model membranes studied by temperature controlled gel filtration chromatography

1997

Biomed. Chromatogr.

View full text Add to dashboard Cite

The use of gel filtration chromatography with Sephadex as a separation medium was used in order to study flunitrazepam (FNTZ) partitioning into artificial model membranes consisting of dipalmitoyl‐phosphatidylcholine (dpPC) vesicles, under controlled temperature conditions. In this system two phenomena are taking place simultaneously: the ligand–liposome interaction and the lipid self‐aggregation to form the liposome. The liposome–FNTZ interaction was evidenced by the non‐enantiography of the first derivative of FNTZ elution peak in frontal chromatography through Sephadex G‐75. On the other hand, the presence of FNTZ reduced liposomes mean size and increased their size dispersion as evidenced by molecular filtration through Sephadex G‐200. The dpPC–buffer FNTZ partition coeficient determined in zonal chromatography through Sephadex G‐10 increased about 33% when the temperature rose above the temperature of dpPC transition from the liquid crystalline to the fluid phase. Gel filtration chromatography seems a suitable technique to study lipid liposome–FNTZ interactions at a qualitative level. In addition, this technique has the advantage over other methods of giving the possibility of observing the mutual effects exerted between the drug and the self‐aggregating structure. © 1997 John Wiley & Sons, Ltd.

show abstract

Evaluation of nonideality from gel chromatographic partition coefficients

Cited by 11 publications

References 36 publications

Thermodynamic nonideality in macromolecular solutions

Thermodynamic nonideality in macromolecular solutions

Macromolecular Crowding: Biochemical, Biophysical, and Physiological Consequences

Partitioning of flunitrazepam into model membranes studied by temperature controlled gel filtration chromatography

Contact Info

Product

Resources

About