The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase.
The metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) has been used at pH 7.0 to monitor the mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase and Zn2+ uptake by regulatory dimers upon displacement of the mercurial reagent with 2-mercaptoethanol. The release of Zn2+ (as reflected by a yellow to orange color change in PAR solutions) is linked to dissociation of the enzyme since the six Zn2+ bonding domains stabilize catalytic and regulatory chain contacts; the rebinding of Zn2+ produces enzyme assembly and a corresponding decrease in the amount of PAR-Zn2+ complex. Using greater than 10-fold PAR to free Zn2+ at pH 7.0, delta epsilon = 6.6 +/- 0.2 X 10(4) M-1 cm-1 at 500 nm (20 degrees C) for (PAR)2Zn2+ complex formation (beta'2 approximately equal to 10(12) M-1). In kinetic studies at pH 7.0, PAR (10(-4) M) has been used to measure the instantaneous concentration of Zn2+ released from micromolar quantities of protein; second-order k = 2 X 10(7) M-1 s-1 for forming the 1:1 PAR:Zn2+ complex. These properties of PAR-Zn2+ interactions make PAR a generally useful reagent for studying Zn2+ release from proteins.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology*; D012118:Resorcinols; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000032:analysis*
D012118:Resorcinols; D001221:Aspartate Carbamoyltransferase; D015032:Zinc; C028398:4-(2-pyridylazo)resorcinol