Light Scattering in Solutions of Serum Albumin: Effects of Charge and Ionic Strength<sup>1,2</sup>

Edsall, John T.; Edelhoch, Harold; Lontie, René; Morrison, Peter

doi:10.1021/ja01166a085

Cited by 183 publications

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“…The molecular weight found for bovine serum albumin in 0.1 A4 socli~~m chloride (pH 5.4), 77,000-79,000, is in good agreement with values found in other light-scattering studies (9,10,13). In Fig.…”

Section: Resultssupporting

confidence: 90%

LIGHT-SCATTERING AND SEDIMENTATION STUDIES OF BOVINE SERUM ALBUMIN AT LOW pH

Reichmann¹,

Charlwood²

1954

Can. J. Chem.

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Light-scattering measurenlents of bovine serum albumin made a t p H 1.9 in 0.1 M-0.45 M potassium chloride show that the molecule is not dissociated, but has the same molecular weight as in neutral solution. At p H 1.9 in the absence of salt aggregation occurs, the extent increasing with time. The sedimentation constant a t pH 1.9 increases from 3.2s in 0.1 n/f potassium chloride to 3.6 in the 0.5 salt, compared with 4.3 in neutral solution. These differences are ascribed to changes of molecular shape. INTRODUCTIONThe kinetic unit of a protein dissolved in aqueous salt solutions is probably an ordered entity, and not merely a randomly coiled polypeptide chain. As Pauling and collaborators (16,19) .have pointed out, the stability of the structure inay be ascribed largely to the cumulative strength of the hydrogen bonds between the carbonyl groups of some peptide links and the amino groups of others, although certain covalent cross-links may also make a contribution to the rigidity. Conditions which promote the breaking of hydrogen bonds could cause changes of size or shape, or even dissociation. The hemoglobin molecule has been shown to dissociate in urea solution (4) and that of insulin a t acid p H (18).Dissociation would be precluded for a protein molecule composed of a single polypeptide chain. End group determinations with bovine serum albumin have revealed a single a-amino terminal group per molecule (26,27), suggesting a single chain. Recent viscosity and optical rotation studies (32) have been taken to indicate an isotropic swelling of the albumin molecule a t acid pH, but no dissociation. On the other hand, Weber (29, 30) originally interpreted his results on the polarization of fluorescence of labelled molecules as demonstrating dissociation under acid (or alkaline) conditions. T o decide between these conflicting views, the weight average molecular weight of bovine serum albumin a t acid pH was determined by light-scattering observations a t various salt concentrations, supplementary information being obtained from sedimentation measurements. MATERIALS AND METHODSThe bovine serum albumin was supplied by Armour and Co. (Lot R370. 295B). The protein solutions were prepared by diluting aqueous solutions with the appropriate solvent, and dialyzing against several changes of solvent. Solvents were prepared by adding concentrated hydrochloric acid to salt solutions. The p H of both solvent and solution was checked in a Becknlan l~l!fanuscript

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Section: Resultssupporting

confidence: 90%

LIGHT-SCATTERING AND SEDIMENTATION STUDIES OF BOVINE SERUM ALBUMIN AT LOW pH

Reichmann¹,

Charlwood²

1954

Can. J. Chem.

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“…In the following section, we present the heretofore unpublished data of Edsall et al 3 and demonstrate that the dependence of light scattering on BSA concentration in 0.15M NaCl and pH values between 4.40 and 7.60 may be quantitatively accounted for over the entire range of BSA concentrations up to 90 g/L by a model in which the BSA molecule is formally represented by an effective hard sphere. In order to account for variations in scattering with pH, it is necessary to permit the diameter of the effective hard sphere to vary with pH.…”

Section: Introductionmentioning

confidence: 70%

Light scattering of bovine serum albumin solutions: Extension of the hard particle model to allow for electrostatic repulsion

Minton

Edelhoch

1982

Biopolymers

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SynopsisThe light scattering of bovine serum albumin (BSA) has been measured at protein concentration up to 90 g/L and at pH values between 4.4 and 7.6. The dependence of scattering on both protein concentration and pH may be quantitatively accounted for by a simple extension of the hard-sphere model for protein solutions [Ross, P. D. & Minton, A. P. (1977) J. Mol. Biol. 112,4374521 allowing for electrostatic repulsions between molecules. According to the extended model, the radius of the effective hard spherical particle representing BSA varies with the net electrical charge of the BSA molecule in a manner which may be calculated from electrostatic theory.

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“…Almost all the experimental points for each solution fall on a straight line. The slopes of most straight lines were expected to be close to zero, since for an isoionic protein or for a protein solution at ionic strength 0.15, the slope is usually found to be near zero (18). Although the slopes were not exactly zero as shown in Fig.…”

Section: Dialyzerl Bovine Serum Albumin Sol~rfionsmentioning

confidence: 86%

On the Aggregation of Bovine Serum Albumin in 0.1 M Salt Solutions

Rosario¹,

Sun²

1973

Can. J. Chem.

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The molecular weight of bovine serum albumin at neutral p H in 0.1 M salt solutions was determined with light scattering measurements. Simultaneously, preferential binding of a salt to the protein was calculated. It was found that the protein aggregates in KI and KSCN, but not in KCI, LiCI, and LiBr solutions. Analysis of the sedimentation behavior has confirmed aggregation as a molecular phenomenon, not an artifact.Le poids molCculaire de serum d'albumine de boeuf a etC determine par diffusion de la lumiere a p H neutre dans des solutions contenant 0.1 M de sel. Simultanement on a calcult I'attachement preferentiel d'un sel a la proteine. On a trouve que la proteine forme des aggrkgats dans ie K I et e KSCN mais pas des solutions de KCI, LiCl et LiBr. L'analyse des patrons de sedimentation a confirme que I'aggregation est un phtnomene moleculaire et non un artefact.[Traduit par le journal]Can. J. Chem. 51, 3781 (1973) Bovine serum album~n aggregates in 0.1 M and the refractive index increments are measured on solu- transition, albumin exists in a compact form, ponent systkm (water-1, protein-2. and salt-3), the plot albumin has been assumed to be a nin-associat: ing protein. For example, the studies of the binding of C1-, I-, and SCN-to albumin (4-6) all ignored the aggregation. In this paper we shall demonstrate that at neutral p H (ca. 5.2) albumin also forms aggregates in the presence of some though not all salts. The molecular weight of albumin at neutral pH, ionic strength 0.10 and room temperature was determined and the preferential binding of each salt to the protein was compared, using the technique of light scattering. Supplementary information about aggregation was obtained with sedimentation measurements. Experimental MaterialsArmour crystalline bovine serum albumin, Lot Nos. A 69908, F 71601, and D 71209, was deionized by Dintzis' method (7). The dimer content varied from 1% to 5% depending upon the lot as judged from sedimentation velocity analyses. N o significant differences were detected in light scattering experiments whichever lot was used.The salts used, KCI, KI, and KSCN were Baker analyzed reagents, while LiCl and LiBr were Matheson, Coleman and Bell reagent grade. Glass-distilled water was used in all the experiments. Light ScatteringAdvantage was taken on the theory of light scattering in multi-component systems that if the light scattering of HC~IAT vs. C2 would give the true molecu'l.ar weight M2, H being the Debye parameter, C2 the concentration in g/ml and AT theexcess turbidity of thesolution over the solvent. If the solutions are not at constant chemical potential but at constant concentration of the solvent system, then the plot of HC2/Ar us. C2 can only give M2', the apparent molecular weight of component 2 (protein), not the true molecular weight. Nevertheless the relation between M2 and M 2 ' enables one t o calculate a quantity (ag3/agZ)7,,,,,,,, which is the preferential binding of the number of grams of the salt to one gram of the protein on molar or molal basis, g being t...

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Light Scattering in Solutions of Serum Albumin: Effects of Charge and Ionic Strength^1,2

Cited by 183 publications

References 0 publications

LIGHT-SCATTERING AND SEDIMENTATION STUDIES OF BOVINE SERUM ALBUMIN AT LOW pH

LIGHT-SCATTERING AND SEDIMENTATION STUDIES OF BOVINE SERUM ALBUMIN AT LOW pH

Light scattering of bovine serum albumin solutions: Extension of the hard particle model to allow for electrostatic repulsion

On the Aggregation of Bovine Serum Albumin in 0.1 M Salt Solutions

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Light Scattering in Solutions of Serum Albumin: Effects of Charge and Ionic Strength1,2

Cited by 183 publications

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LIGHT-SCATTERING AND SEDIMENTATION STUDIES OF BOVINE SERUM ALBUMIN AT LOW pH

LIGHT-SCATTERING AND SEDIMENTATION STUDIES OF BOVINE SERUM ALBUMIN AT LOW pH

Light scattering of bovine serum albumin solutions: Extension of the hard particle model to allow for electrostatic repulsion

On the Aggregation of Bovine Serum Albumin in 0.1 M Salt Solutions

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Light Scattering in Solutions of Serum Albumin: Effects of Charge and Ionic Strength^1,2