ENDOPOLYGALACTURONASE GOLD COMPLEX - PARAMETERS INFLUENCING THE LABELING OF PECTIC ACID IN THE CELL-WALLS OF FLAX HYPOCOTYL

An enzyme/colloidal-gold complex was prepared using a purified endopol ygalacturonase (endoPGH, E.C. 3.2.1.15). Gold particles with a radius of 4.8 nm were prepared. To stabilize the gold particles, it was neces sary to add a minimal amount of enzyme (approximately 0.5 mug) at a pH higher than its isoelectric pH. Under these conditions, the number of protein molecules adsorbed per gold particle was approximately one. T he parameters influencing the diffusion of the endoPGH-gold complex to its target molecules were screened and tested through quantitative ap proaches: (1) the labelling efficiency q was calculated according to t he equation for the diffusion of gold markers from the bulk solutions to target molecules and (ii) the labelling density N/S was measured by counting gold particles on electron micrographs. The highest value of N/S was obtained by determining the optimal values for the parameters outlined in the equation for q. To obtain the highest value of N/S, i t was not necessary to choose values for these parameters which gave h igh values for q. The specificity of the probe towards homogalacturoni c acid blocks was established through controls. The localization of th ese blocks was illustrated throughout the walls of different kinds of flax seeding cells.