Figure 3. The reaction of rEP18 with hydrazide-agarose in the presence or absence of aniline.

Hydrazide-agarose (200 μl packed resin) was washed with either buffer KP or KP containing 100 mM aniline. To the packed resin was added 200 μl of 20 nM radiolabeled rEP18 reacted in 1 mM NaIO₄ for 30 min at 20°C in the dark. The mixtures were stirred magnetically and a portion removed at the times shown. The resin was washed twice with 5 volumes of 1 M NaCl, 50 mM potassium phosphate, pH 6.8 and once with 5 volumes of SDS-PAGE sample buffer. The combined washes and the washed resin were then counted in a scintillation counter without scintillation fluid (Cerenkov radiation). The total counts per minute (washes plus resin) and the counts per minute of the resin were then used to calculate the percentage of the total counts per min coupled to the resin. The results from the semi-logarithmic plot (data not shown) gave a straight line consistent with an apparent first-order reaction with a half-time of 27 min in the absence (squares, grey line) and 23 min in the presence of aniline (triangles, black line).