Lysozyme association constants, determination

Two different sequential saturation assays are modified from the ELISA25 to determine association constants of antibody with lysozymes. In both assays, a constant antibody concentration is incubated with varying concentrations of lysozyme until equilibrium is reached. The time necessary must be determined empirically for each antibody for the antibody HyHEL-10, this was determined to be at least 16 hr,26 and all incubations were performed for 16-24 hr. At the end of the incubation, an aliquot of the mixture is treated with an excess of reagents to sample free (unbound) antibody combining sites by incubating with labeled or solid-phase coupled... 

Some 13 years after Carr s original observations, Kretsinger (1976), in his review of calcium-binding proteins, assumed that lysozyme can attach Ca(ll), as well as other cations. It was not until 1981 that binding of Ca(Il) to lysozyme was further studied. Imoto et al. (1981) determined the stability (association) constant (40 M" ) and found that lysozyme is inhibited in the presence of Ca(II), showing only 26% of the activity of the free enzyme toward hexa-AT-acetylglucosamine. Because of this inhibition, they predicted that Ca(II) binds near the catalytic carboxyls. Furthermore, Ca(II) shifts the native-denatured transition in lysozyme toward the native state, and thus has some preservative effect on the protein. 

Specific binding of the competitive inhibitor N-acetyl-D-glucosamine to lysozyme modified the CD spectrum in the range 240—300 nm, which indicates changes in the orientation of the aromatic amino acid residues at the binding site (363). Association constants were determined from the... 

The association constant for the binding of Gd + ions to lysozyme has been determined at pH 5.2 (by measuring the release of protons) and at pH 6.5 (by u.v. spectroscopy). In agreement with X-ray data, it was found that Gd + ions bind to the carboxy-groups of L-glutamic acid-35 and L-aspartic acid-52 at the junction of sites D and E in the cleft of the enzyme. The association constants for the binding of several inhibitors and substrates to lysozyme and to the complex with Gd + were determined by fluorescence spectroscopy the lactone derived from the tetra-iV-acetylchitotetraose (a transition-state analogue) had a... 

The ionization constant of L-histidine 15 of hen eg -white lysozyme, determined by H n.m.r. spectroscopy, exhibited a dependence upon concentration, whereas the values of this residue and of ester 35, 108 lysozyme were observed to be almost independent of protein concentration. The L-tryptophan residues of lysozyme were examined by C n.m.r. spectroscopy as functions of pH, protein concentration, and temperature. On the basis of the results of these investigations, it was proposed that the portion of the lysozyme surface involved in head-to-tail association includes L-histidine 15... 

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