Elbert A. Peterson

[1] Column chromatography of proteins: Substituted celluloses

Publisher Summary Chromatography of proteins on cellulose ion exchangers involves primarily the establishment of multiple electrostatic bonds between charged sites on the surface of the adsorbent and sites bearing the opposite charge on the surface of the protein molecule. The number of such bonds that can be established determines the concentration of competing ions required for the release of the bound molecule. Thus, proteins differing significantly in charge density, or in number of charges by virtue of size, may be expected to differ in their requirements for elution. Charge distribution can also be regarded as a factor. But it is the total effect of these factors that determines the affinity of the protein for the adsorbent, so a simple relation between any one of them and the chromatographic behavior of the protein in question is not always obtain. The situation is further modified by the possibility that in some cases nonelectrostatic forces might play an important role.

Some experimental factors in the gradient chromatography of serum proteins.

Abstract A procedure is described for the chromatographic scanning of protein mixtures such as that in serum, using a single, compound gradient of increasing molarity and decreasing pH, adjustable in shape to suit the requirements of a given experiment. The effects of changes in the gradient shape and volume on the appearance of chromatographic components in human serum are demonstrated. Flow rates were varied over a wide range without much effect; the upper limit was determined largely by the mechanical properties of the adsorbent rather than by the adsorption process itself. With one exception, the serum proteins emerged at their original positions when rechromatographed. Anomalous behavior of albumin in this regard was found to be caused by contact with a contaminant in the dialysis tubing, and a similar effect was obtained when serum was treated with oleic acid. Several buffer systems are compared with respect to eluting power and the resolution attainable.

Ion-exchange displacement chromatography of proteins, using narrow-range carboxymethyldextrans and a new index of affinity

A new method of characterizing carboxymethyldextrans with respect to their relative affinities for an anion exchanger can be applied not only to pure preparations but also to fractions of a displacement chromatogram in which these polyanions have been used as spacers. This, along with the development of a means of preparing carboxymethyldextrans with narrow ranges of affinity, as contrasted with the very heterogeneous preparations previously used, has greatly facilitated the focusing of resolving power on critical regions of the chromatogram. The effectiveness of this approach is illustrated by the separation of proteins that differ only slightly in affinity, using the genetic variants of beta-lactoglobulin as one model and the numerous components of purified ovalbumin as another.

Ion-exchange displacement chromatography of serum proteins, using carboxymethyldextrans as displacers

Abstract A system of displacers comprising carboxymethyldextrans with progressively higher content of carboxyl groups forms a displacement train in which absorbed proteins find positions according to their affinities for the adsorbent, an anion exchanger. Because little or no salt need be used, effluent fractions can be evaluated directly by gel electrophoresis. Application to the fractionation of serum proteins is demonstrated.

The effect of mercaptoethanol and urea on the molecular weight of hemoglobin

Abstract 1. 1. Unlike horse hemoglobin, human hemoglobin is not dissociated by concentrated urea, although its shape is considerably altered. 2. 2. Horse and human hemoglobins are equally dissociated by mercaptoethanol, and even further, but still equally, by a combination of mercaptoethanol and urea. 3. 3. The mechanism of mercaptoethanol action is not clear, but the data suggest that it attacks bonds other than those sensitive to urea. 4. 4. Sedimentation-diffusion measurements, in this study, indicate that dissociation of horse hemoglobin by concentrated urea is less complete than that found in previous reports. An explanation in terms of heterogeneity and a rapid dissociation-association equilibrium is suggested.

Preparative high-performance liquid chromatography of proteins on an anion exchanger using unfractionated carboxymethydextran displacers

It has been previously demonstrated that carboxymethyldextran (CMD) displacers fractionated on an ion-exchange column can be used for the resolution and high capacity displacement chromatography of proteins on high-performance ion-exchange columns. It is shown, in this manuscript, that high resolution and high capacity are obtainable using unfractionated CMD displacers. Up to 25 mg of a mixture of ovalbumin, alpha-lactalbumin, and soy-bean trypsin inhibitor along with many impurities were separated on a 3.3-ml Altex DEAE-5PW column using three unfractionated CMD displacers and a final CMD displacer. A method for removing the displacers from proteins, on a hydrophobic interaction column, is also described.

Experimental problems in the use of commercially prepared DEAE-celluloses for protein chromatography

Abstract Commercially available DEAE-celluloses have been tested in a highly standardized system and found to vary beyond tolerable limits in their physical and chromatographic properties. A simple test is described which makes it possible to define the general properties of a commercial adsorbent without carrying out a full gradient elution.

Purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers

The anion-exchange separation of complex protein mixtures by displacement chromatography using spacer displacers driven by high-affinity final displacers is demonstrated. Guinea pig serum was separated on a medium-resolution adsorbent using a single heterogeneous mixture of carboxymethyldextran displacers to space the protein components. Mouse liver cytosol was separated on a low-resolution adsorbent using six carboxymethyldextran spacer displacers of increasing column affinity. The demonstration of the purification of alkaline phosphatase from E. coli periplasm by displacement chromatography on a high-performance liquid chromatography column is reviewed. The benefits of spacer displacers for separating minor components from complex biological protein mixtures is discussed. A simplified method for preparing carboxymethyldextran displacers is presented.

Purification of Gc-2 globulin from human serum by displacement chromatography: A model for the isolation of marker proteins identified by two-dimensional electrophoresis

A protein that was initially known only as a minor spot in two-dimensional electrophoresis patterns of serum obtained from certain psoriasis patients, particularly those with a pustular component to their disease, has been purified by two stages of ion-exchange displacement chromatography on DEAE-Sephacel at different pH levels, followed by elution chromatography on hydroxylapatite. The purification was followed by examining the column fractions directly by two-dimensional gel electrophoresis. The capacity of the displacement system, which utilized carboxymethyldextrans as displacers, was very high; 6 ml of dialyzed serum applied to a 7-ml column in the initial stage resulted in a very substantial enrichment of the target protein. The second displacement stage yielded a highly purified product, contaminated only by A-1 lipoprotein. The latter was removed by hydroxylapatite chromatography. The purified protein was subsequently identified as Gc-2 globulin, a vitamin D-binding protein, by immunological procedures. The results demonstrate the effectiveness of ion-exchange displacement chromatography in focusing resolving power on the relatively narrow range of affinities represented by the target protein and its immediate neighbors in a chromatogram, as well as the applicability of the system to the isolation of a protein known only by its position in a two-dimensional electrophoretic pattern.

Parative high-performance liquid chromatography of proteins on ion-exchange columns with carboxymethyl dextrans as displacers

Abstract It is shown that carboxymethyldextrans can be used to displace proteins from high-performance ion-exchange columns. The high resolving power of the method is demonstrated by the total separation of the A and B genetic variants of the β-lactoglobulins, which have only 0.1 pH difference in their isoelectric points. The separation of up to 12.8 mg of the β-lactoglobulins on a 150-μl column demonstrates the potential for high capacity in displacement chromatography.