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Clinical Chemistry 21: 591-594, 1975;
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Clinical Chemistry, Vol 21, 591-594, Copyright © 1975 by the American Association for Clinical Chemistry

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

Hiroshi Kawaguchi 1 and David S. Auld 1

1 Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School; and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Mass. 02115.

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.


Key Words: microwave spectrometry • gel exclusion chromatography • reverse transcriptase • cancer • virus • trace elements • microscale analysis • • microwave-induced emission spectrometry • woolly monkey • DNA polymerase

Submitted on December 2, 1974
Accepted on January 29, 1975






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Copyright © 1975 by the American Association for Clinical Chemistry.