LABELLING OF CATALYTIC SITE OF LYSOZYME

A method for direct measurements of the binding of N-acetyl glucosamine polymers to the catalytic site of lysozyme is described. The method utilizes the dye Biebrich scarlet for which the enzyme has a single binding site with a dissociation constant of 0.3 mM. The data indicate that the trimer and hexamer of N-acetyl glucosamine do not displace the dye from the unproductive binding site of lysozyme to which they both bind with a dissociation constant of about 10 μM. The two sugars competitively displace the dye from the catalytic site of the enzyme, allowing one to differentiate between productive and unproductive binding of these two substrates. Spectrophotometric determinations of the concentrations of the enzyme-dye complex in the presence of various concentrations of substrate gave productive binding dissociation constants for the trimer and hexamer of 20 mM and 5 μM respectively. © 1969.