Microanalytical System for Determination of Picogram Quantities of Metals in Metalloenzymes, as Illustrated with Zinc-containing Enzymes

Abstract

Abstract Microwave-induced emission spectrometry combined with gel-exclusion chromatography provides a microanalytical system capable of precisely measuring 10-10 to 10-13 g of metal in microgram amounts of enzyme. Such sensitivity greatly exceeds that of other, more conventional methods. Metal quenching agents and low-molecular-weight protein contaminants were removed from the enzyme by Sephadex G-100 chromatography in microbore columns (0.3 x 25 cm). Droplet fractions were analyzed for zinc by the present method, for enzyme activity, and for protein content. With this analytical system we could demonstrate that stoichiometric amounts of zinc are present in the RNA-dependent DNA polymerase, the reverse transcriptase, from woolly monkey type C RNA tumor virus. The precision of the method for zinc was demonstrated by the coefficient of variation of 4.4% for 10 µg of zinc per liter. Validity and accuracy of the method were established by determining zinc in a series of zinc metalloenzymes of known metal content and stoichiometry.