Binding constants of levans and D-fructo-oligosaccharides to BALB/c and NZB D-fructan-specific, myeloma proteins, determined by affinity electrophoresis

Abstract

The association constants for the interaction of BALB/c (UPC 10, Y5476, W3082, and UPC 61) and NZB (PC 3660) myeloma anti- D-fructans in pure form, or in ascitic fluids, with high-molecular-weight levans (K a) and with such low-molecular-weight compounds as rye-grass levan, inulin, sucrose, and D-fructo-oligosaccharides ▪ were determined by affinity electrophoresis, measuring the extent of retardation of the D-fructan-specific band by levan and its restoration by the low-molecular-weight compounds and oligosaccharide haptens. With different levans, K a values ranged from 1.14 x 10 5 to 1.52 x 10 6 mL/g for PC 3660, 1.35 x 10 5 to 6.45 x 10 5 mL/g for UPC 10, 1.0 x 10 4 to 7.9 x 10 4 mL/g for Y5476, 7.63 x 10 3 to 6.38 x 10 4 mL/g for W3082, and 3.35 x 10 3 to 1.54 x 10 4 mL/g for UPC 61. The retarded, D-fructan-specific bands of W3082 and UPC 61 were restored by inulin, having β- D-(2→1)-linkages, and rye-grass levan, having β- D-(2→6)-linkages, and those of PC 3660, UPC 10, and Y5476 by rye-grass levan. The ▪ values of inulin with W3082 and UPC 61 were 10 times those of rye-grass levan. The ▪ values of inulin (3.65 x 10 5 M -1 for W3082, and 4.44 x l0 5 M -1 for UPC 61) were very similar to those of [β- D-Fru f-(2→1)] 2-β- D-Fru f-(2→6)- D-Glc ( 3) (3.95 x 10 5 M -1 for W3082, and 4.5 x 10 5 M -1 for UPC 61). With sucrose and the D-fructo-oligosaccharides, the order of ▪ values of W3082 and UPC 61 was 3 > β- D-Fru f-(2→1)-β- D-Fru f-(2→6)- D-Glc > sucrose > β- D-Fru f-(2→6)- D-Glc. With rye-grass levan, ▪ values were 4.25 x 10 5 M -1 for PC 3660, 2.7 x 10 5 M -1 for UPC 10, and 8.77 x 10 4 M -1 for Y5476. These results confirm earlier findings that W3082 and UPC 61 have dual specificity for β-(2→1) and β(2→6) D-fructofuranosyl linkages, that PC 3660, Y 5476, and UPC 10 have specificity for β-(2→6) D-fructofuranosyl linkages, and that the combining sites of W3082 and UPC 61 are most complementary to the tetrasaccharide 3. That W3082 and Y5476 share the same, cross-reacting idiotype, although their combining sites differ in specificity, provides further evidence that these two properties do not run parallel.