A test to evaluate the effect of individual components of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffers on enzymatic activity.

A simple dilution test for evaluating the individual effect on enzymatic activity of [Ca2+], [EGTA], or [Ca.EGTA] variations in Ca-EGTA buffers is presented. We verified that a 50-fold dilution of the buffer (25-0.5 mM) at constant pH did not affect [Ca2+] (measured with fura-2), whereas [EGTA] and [Ca.EGTA] varied. Therefore the test can be applied to evaluate the proper effect of Ca2+ in a Ca-EGTA buffer on enzyme activity because such an effect is expected to remain unchanged upon dilution of the buffer. Applications of the test are shown for three enzymes apparently sensitive to Ca2+ but found to be effectively influenced only by Ca.EGTA (liver glucose-6-phosphatase), EGTA (intestinal mucosa phosphatase), or indeed Ca2+ (brain cyclic nucleotide phosphodiesterase).