Chromatographic Properties of Gamma Globulin: Behavior of Serum Gamma Macroglobulins*

Abstract

The cellulose ion exchangers (1) have proved to be useful chromatographic media for the serum proteins. In the fractionation of normal human serum on the anion exchanger, diethylaminoethyl (DEAE) cellulose, application of buffer gradients of increasing concentration and decreasing pH results in the appearance of components in the effluent fluid in order of increasing electrophoretic mobility (2), indicating that ion exchange is the predominant mechanism involved. In the case of the gamma globulin component, some subfractionation was demonstrated in terms of progressively increasing electrophoretic mobility of column effluents of this fraction. Little attention has been directed, however, to the chromatographic properties of the gamma globulins of high molecular weight, 19S or greater, which are found in large quantities in certain pathological sera, and also in small quantities in normal human sera (3).In the course of anion exchange fractionation of sera from patients with rheumatoid arthritis, the "rheumatoid factor" (4-6), a gamma globulin constituent of high molecular weight (7), appeared in column effluents far removed from the bulk of the gamma globulin (8). This observation indicated that the chromatographic behavior of certain gamma globulins, and possibly of other serum proteins, on DEAE cellulose is governed not only by ion exchange but also by other factors which depend in part upon molecular size. Our attention was therefore directed to sera known to buffer at pH 7. The equilibrated exchanger was then poured onto columns (2.5 X 15 cm) equipped with fritted glass filters, and packed under mild air pressure. Before chromatogarphy, sera were diluted with an equal volume of distilled water and dialyzed for two days against 3 to 6 L of 0.01 M phosphate buffer at pH 8.5 to 9. In one instance, the euglobulin precipitate from the serum of a patient with macroglobulinemia was fractionated. This was prepared by 15-fold dilution of 4 ml of the serum with cold water. The precipitate formed was harvested by centrifugation, washed four times with cold water, dissolved in 0.01 M phosphate buffer at pH 8.6 and chromatographed.Chromatographic fractionation was carried out at room temperature using relatively rapid flow rates of 1 to 2 ml per minute as described previously (8). The protein content of each effluent fraction was determined by measurement of ultraviolet absorption at 280 m/u in the Beckman spectrophotometer. Readings were taken as fractions and were collected in order to follow the course of separation and to determine when a change in the eluting buffer was required. At the end of each chromatographic experiment, samples of each peak were concentrated 10-to 100-fold by ultrafiltration through Mies collodion bags 2 to a final protein concentration of approximately 1 per cent. Paper electrophoretic analysis was carried out in a Spinco (Durrum) apparatus at pH 8.6 (r/2 = 0.075) for 16 hours at 120 v and 4 ma. After staining with bromphenol blue, paper strips were analyzed in a densitometer.3 Ultracentrifug...

The Formation of Macroglobulin Antibodies. I. Studies on Adult Humans*

“…The fractions comprising each peak were pooled and tested for antibodies to typhoid 0, typhoid H, and paratyphoids A and B by standard agglutination procedures (10). Antibody activities associated with the first (pH 7.0, 0.01 M) and the last (pH 5.0, 0.3 M) chromatographic peaks were considered to contain 7S and 19S -y-globulins, respectively, on the basis of the behavior of these proteins on DEAE-cellulose (9,11,12). Although the first, or 7S, peak contains no 19S globulins, the last peak is contaminated by 7S protein, which could account for part of the antibody titers associated with this fraction.…”

The Formation of Macroglobulin Antibodies. Ii. Studies on Neonatal Infants and Older Children*

“…After dialysis, sera were stored at -20°C. The dialyzed sera, usually 1 to 3 ml, were fractionated by column chromatography on diethylaminoethyl (DEAE) -cellulose as previously reported (10,11). Proteins were eluted with phosphate buffers of increasing concentration and decreasing pH.…”

Cathepsin D Agglutinators in Rheumatoid Arthritis

“…The hemagglutination inhibition tests were carried out with high-titer RA serum as previously described (4). To determine the type of the agglutinators, some rheumatoid sera were chromatographed through DEAE cellulose columns by a stepwise elution method using increasing salt concentrations and decreasing pH in the buffer as described by LoSpalluto ef a1 (15) and Smiley et a1 (16).…”

“…Three serum samples from patients with seropositive RA containing relatively high titers of CDA according to Figure 1 (reciprocal titer: 64) and one serum sample from a patient with seronegative RA (reciprocal CDA titer: 32) were chromatographed through DEAE columns by the stepwise elution method described by LoSpalluto et a1 (15) and Smiley et al (16). Four peaks were recovered; peak I contained much IgG and peak IV contained much IgM and very little or no IgG and IgA.…”