Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy.

A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv "Sparkle") by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O(2) concentration can be calculated. With this method, we determined internal O(2) while measuring nitrogenase activity (C(2)H(2)) in detached pea nodules over a range of external O(2) concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O(2) concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C(2)H(2)) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.