Jack bean urease (EC 3.5.1.5). I. A simple dry ashing procedure for the microdetermination of trace metals in proteins. The nickel content of urease.
A simple and inexpensive procedure for determination of microgram quantities of metal ions in proteins is described and tested with nickel and iron. The method involves (a) dry ashing in an oxygen atmosphere at 450-460 degrees C in Pyrex vessels, (b) conversion of the metal oxides or other compounds to readily soluble species, and (c) spectrophotometric analysis. An improved procedure for the direct spectrophotometric determination of nickel using dimethylglyoxime is accurate to +/- 2% or better with samples of 1-5 microgram of nickel. These techniques were used to determine that the nickel content of freshly prepared jack bean urease in 2.00 +/- 0.12 g-at./96 600 g protein. The corresponds to 2.0 nickel ions per subunit. This result was confirmed by atomic absorption analysis, which also showed that calcium, manganese, cobalt, and iron are not present in significant amounts in urease.