BINDING OF RETINOIC ACID TO BETA-LACTOGLOBULIN VARIANT-A AND VARIANT-B - EFFECT OF PEPTIC AND TRYPTIC DIGESTION ON THE PROTEIN LIGAND COMPLEX

Studies were undertaken to evaluate kinetic parameters, such as appare nt dissociation constant (Kd) and protein/ligand molar binding ratio ( n) for beta-lactoglobulin variants A and with B retinoic acid. In addi tion, the effect of digestive enzymes such as pepsin and trypsin on th e protein and bound complex was also investigated. Based on fluorometr ic quenching experiments beta-lactoglobulin is able to bind retinoic a cid. The molar binding ratio for the protein/ligand interaction is dep endent on the method used to evaluate the kinetic parameters, is. dire ct linear plot (18) vs. regression analysis (7, 17). it was concluded that the direct linear plot of EISENTHAL and CORNISH-BOWDEN (18) is mo re appropriate, as it deals with the data in observation space elimina ting transformation errors. According to the direct linear graphs the molar binding ratio for protein to ligand (n) was 1: 1.1, which indica tes that 1 molecule of protein bind 1 molecule of retinoic acid. In ad dition, the apparent dissociation constants were dependent on the anal ytical interpretation with the direct linear plot giving affinities, w h ich were closer to other published data, i.e. Kd = 1.96 x 10(-7) for beta-lactoglobulin variant A and 1.75 x 10(-7) for variant B. The ret inoic bound beta-lactoglobulin was examined in terms of its ability to withstand peptic and tryptic digestion at 37 degrees C. It was conclu ded that pepsin held very little significance in terms of digestive pr ocesses. beta-lactoglobulin was highly susceptible to digestion by try psin (in excess of 90% was digested). When beta-lactoglobulin was boun d to retinoic acid there was a significant reduction in its digestibil ity (less than 25% of the protein was broken down) indicating that hyd rophobic molecules such as retinoic acid may serve to protect beta-lac toglobulin from specific digestive processes.