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 endopolygalacturonase (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 necessary 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. The parameters influencing the diffusion of the endoPGH-gold complex to its target molecules were screened and tested through quantitative approaches: (1) the labelling efficiency q was calculated according to the 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, it was not necessary to choose values for these parameters which gave high values for q. The specificity of the probe towards homogalacturonic acid blocks was established through controls. The localization of these blocks was illustrated throughout the walls of different kinds of flax seeding cells.