In polyacrylamide electrophoresis, mobilities of dextran-specific myeloma proteins W3129 and QUPC52 were retarded when dextrans were added to the separating gel. Retardation by dextran was reversed by adding isomaltose oligosaccharides to the gel. From measurements of the extent of retardation by dextran or of reversal by hapten as a function of concentration, association constants of dextrans (Ka) and of oligosaccharides (Kia) were determined.
With W3129, NRRL dextrans B 742 FR.S, B 742 FR.L, N279, B 1355 FR.S, B 1399, B 1299 FR.S, and NRC fractions 6, 7, and 8 have Kα values ranging from 2 to 6 × 104 ml/g. With QUPC52, dextrans in general show lower Ka than with W3129; N279, NRC fractions 6, 7, and 8, and B 1399 have Ka values ranging from 1 to 7 × 104 ml/g, whereas B-742 FR.S, B-1299 FR.S, and B-1355 FR.S have Ka values ranging from 3 to 6 × 102 ml/g. Dextran D3, a completely linear α(1 → 6)-linked glucosyl chain having a m.w. of 36,500 has a relative high affinity for QUPC52 (Ka = 2.3 × 103 ml/g or 8.3 × 104 M-1), although it does not show any affinity for W3129. This result confirms that myeloma protein W3129 is specific for a terminal chain of α(1 → 6)-linked glucosyl residues, whereas QUPC52 reacts with the nonterminal portion of α(1 → 6)-linked glucosyl chains. Kia values obtained coincided with those by other methods reported earlier by Cisar et al. With W3129, affinity increases with chain length up to isomaltopentaose (IM5) with the highest affinity Kia = 6.7 × 104 M-1; Kia of isomaltohexaose (IM6) and isomaltoheptaose (IM7) being 6.3 × 104 and 2.3 × 104 ml/g, respectively. With QUPC52, affinity increases as the number of residues in the glucosyl chain increases, reaching an upper limit with IM6 and IM7, 1.2 and 1.4 × 104 M-1.
This method of affinity electrophoresis is useful for obtaining binding constants ranging from 102 to 106 M-1. It may be performed on antibodies, lectins, and other binding proteins by using not only the purified proteins but also crude protein mixtures such as the ascitic fluid used in this study.