Send to

Choose Destination
See comment in PubMed Commons below
Anal Biochem. 1994 Sep;221(2):379-86.

A continuous, spectrophotometric activity assay for nitrogenase using the reductant titanium(III) citrate.

Author information

Department of Chemistry and Biochemistry, Utah State University, Logan 84322-0300.


A continuous, spectrophotometric assay for determining electron transfer rates through nitrogenase during substrate reduction reactions was developed. The assay takes advantage of the facts that Ti(III) citrate can serve as a reductant for nitrogenase-catalyzed reduction reactions and that oxidation of Ti(III) citrate to Ti(IV) citrate results in a dramatic change in its absorption spectrum. Ti(III) citrate supported nitrogenase-catalyzed substrate (e.g., H+ or acetylene) reduction reactions at about the same rate as that supported by the reductant dithionite (S2O4(2-)). In addition, Ti(III) citrate had an absorption maximum centered at 325 nm, while oxidized Ti(IV) citrate had a much lower absorption in this wavelength region. An absorption coefficient for Ti(III) citrate of 0.73 at 340 nm was determined by titration with redox dyes with known absorption coefficients. Using this experimentally determined absorption coefficient, we developed an assay that provides a convenient way to determine electron transfer rates through nitrogenase in real time by spectrophotometrically following the oxidation of Ti(III) citrate to Ti(IV) citrate. Average electron transfer rates of 3749 +/- 218 nmol of electrons iron protein-1 for H+ reduction were determined using this assay which are directly comparable to the rates calculated from fixed time point, gas chromatographic assays of H2 formation. The utility of the Ti(III) citrate assay for nitrogenase is discussed and demonstrated using the nitrogenase inhibitors MgADP, CN-, and NO.

[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center