CHARACTERIZATION OF GLIADIN-GALACTOMANNAN INCUBATION MIXTURES BY ANALYTICAL ULTRACENTRIFUGATION .1. SEDIMENTATION-VELOCITY

The aim of this work is to examine the possible interaction and extent thereof of the polysaccharide galactomannan (GAL) with the cereal pro tein gliadin (GLI) and a peptic-tryptic degraded gliadin (PT-GLI) by a nalytical ultracentrifugation. The work is part of a series of investi gations into the field of coeliac disease (gluten-induced enteropathy) as gliadins are known to be toxic for patients with this disease. The molecular integrity of the GAL and GLI preparations was first checked by sedimentation velocity and sedimentation equilibrium. Sedimentatio n velocity showed single boundaries indicating homogeneity and low-spe ed sedimentation equilibrium gave plausible apparent weight average mo lar masses of 180,000 g/mol for GAL and 20,000 g/mol for GLI. PT-GLI, GLI and GAL in phosphate buffer (pH 6.5) and the incubated mixtures (s tirred for 3 h at 37 degrees C; PT-GLI:GAL = 3.53:1, wt.wt.; GLI:GAL = 0.23 and 0.55:1, wt.wt.) were then investigated by sedimentation velo city at a temperature of 20 degrees C. The plots of 1/s(20) VS. c Of G AL, PT-GLI-GAL and GLI-GAL mixtures after incubation show a significan tly different shape suggesting the presence of interactions. According to the equation 1/s(20) = 1/s(20)(o)(1+k(s)c), values for (s(20)(o), k(s)) of {(4.02+/-0.23) S, (490.9+/-28.9) ml/g}, ((5.92+/-0.24) S, (11 52+/-44) ml/g} and ((5.38+/-0.19) S, (1141+/-38) ml/g) for GAL and PT- GLI-GAL and GLI-GAL mixtures, respectively, were obtained. The concent ration of GAL ranged from 0.75-3.0 mg/ml for GAL alone and from 0.34-1 .50 mg/ml in the incubated mixtures. This apparent indication for a we ak non-covalent protein-polysaccharide interaction was further support ed by UV absorption spectrometry and gel filtration. Copyright (C) 199 6 Elsevier Science Ltd