Fractions from the column which were to be subjected to analytical ultracentrifugation were concentrated by negative pressure dialysis and dialyzed for 16 hr in the cold against at least 500 volumes of phosphate buffered saline, pH 7.2, ionic strength 0.154. They were then centrifuged at 59780 rpm for 35 to 80 min at 20 ` C in a Spinco model E ultracentrifuge at a protein concentration of 1.00 to 1.25%. Sedimentation coefficients were computed as ** f values and relative amounts of the various components were calculated from the Schlieren patterns.

For preparative ultracentrifugation, fractions from the column were concentrated by negative pressure dialysis to volumes of 1 ml or less, transferred to cellulose tubes and diluted to 12 ml with isotonic saline. Ultracentrifugation was then carried out in a Spinco model L ultracentrifuge at 40000 rpm for 125 to 150 min, refrigeration being used throughout the run. Successive 1 -- ml fractions were then drawn off with a hypodermic syringe, starting at the top of the tube, and tested for agglutinin activity.

Other methods will be described below.

The insoluble material which precipitated during dialysis against starting buffer always showed intense agglutinin activity, regardless of the blood group of the donor. With either of the gradients described, chromatography on DEAE-cellulose separated agglutinins of the ABO series into at least three regions (Figs. 1 and 2): one of extremely low anionic binding capacity, one of low anionic binding capacity and one of high anionic binding capacity. These have been labeled Regions 1, 2, and 4, respectively, in Fig. 1. When the early part of the gradient was flattened, either by using the gradient shown in Fig. 2 or by allowing the ``cone sphere'' gradient to become established more slowly, Region 2 activity could sometimes be separated into two areas (donors P. J. and R. S., Fig. 1 and E. M., Fig. 2). The latter procedure gave rise to a small active protein peak (Region 1a) between Regions 1 and 2. In 2 of 15 experiments on whole serum a region of agglutinin activity with intermediate anionic binding capacity was detected (Region 3, Fig. 1). Moreover, after concentration using negative pressure dialysis, agglutinin activity could sometimes be detected in the region designated 2a (donors P. J., D. A., and J. F., Fig. 1).