Original ContributionReactions of nitrite with hemoglobin measured by membrane inlet mass spectrometry
Introduction
Considerable research has focused on processes intrinsic to erythrocytes and hemoglobin that may play a role in vasoregulatory activity, processes such as the generation of NO from the anion nitrite and the oxylabile S-nitrosylation of cyteine residues [1], [2]. There has been great interest in the reactions of nitrite with hemoglobin since initial reports of the nitrite reductase activity of deoxy-Hb(FeII) [3]. The possible association between nitrite and vasodilator activity has been considered for many years, a topic that is well reviewed [1], [2], [4], [5]. This has stimulated studies to determine whether the interaction of nitrite with red cells produces vasodilation through mechanisms related to the nitrite reductase activity of deoxy-Hb(FeII). Profound issues have arisen over the mechanisms by which nitrite might produce vasodilation. One issue is whether NO is able to escape from the red cell since it is avidly bound up by deoxy-Hb(FeII). This has prompted some suggestions that there are processes near the cell membrane that promote efflux of NO and/or that the nitrite reductase activity of hemoglobin produces intermediates such as N2O3 that efflux the cell and decay to NO and NO2 in the plasma [1], [2], [4].
We demonstrate here the application of membrane inlet mass spectrometry to the reactions of nitrite with hemoglobin. This method is based on the use of semipermeable membranes that allow a flux of uncharged molecules from solution into the ionization chamber of the mass spectrometer. The use of membrane inlet mass spectrometry has been well described in the detection of volatile, uncharged organic solutes in aqueous solution and blood [6], and in the study of the reactions of CO2 and the catalysis by carbonic anhydrase [7]. Although the application of this technology to the measurement of dissolved NO has been reported [8], an application more practicable for biological reactions of NO is needed, one that could possibly also be used in physiological experiments. Accordingly, we report here the use of a membrane inlet system described earlier [9] applied to the study of the reactions of nitrite with hemoglobin.
In this work we demonstrate the type of data that can be obtained investigating the reactions of nitrite and hemoglobin while directly detecting nitric oxide in solution. Although the nitrite reductase activity of hemoglobin produces micromolar amounts of NO under our conditions, much of this NO is not detected since it is tightly bound to deoxy-Hb(FeII). Membrane inlet mass spectrometry detects nanomolar levels of NO on addition of 8 mM nitrite to 38 µM deoxy-Hb(FeII). Moreover, this is observed starting several minutes after the reaction of nitrite and hemoglobin begins. The addition of nitrite to oxy-Hb(FeII) and met-Hb(FeIII) did not generate free, unbound NO over the 25 min time course of these experiments. Although addition of cyanide to bind met-Hb(FeIII) did not alter the accumulation of free NO caused by reactions of nitrite with deoxy-Hb(FeII), addition of mercuric ions to bind thiol and possibly imidazole groups increased the rate and maximal level of the accumulation of NO. These reactions have been extensively studied by many methods over the years, and the membrane inlet mass spectrometric method provides a rather unique view.
Section snippets
Materials
Human hemoglobin was purified from human red cells and also obtained from Sigma-Aldrich. That obtained from human red cells was purified by lysing cells with water and centrifuging to remove membranes. The resulting lysate was treated with an affinity gel (p-aminomethylbenzenesulfonamide-agarose, Sigma) to remove carbonic anhydrase. Hemoglobin from Sigma was nearly entirely in the form of met-Hb(FeIII) and was reduced to the ferrous form using dithionite. The resulting solution was passed
Response of the membrane inlet mass spectrometer
Fig. 2 shows typical ion currents obtained with the membrane inlet immersed in a solution containing 38 µM deoxy-Hb(FeII) at pH 6.8. At time zero, sodium nitrite was added to an initial concentration of 8 mM. The data show the ion currents at mass-to-charge ratios of m/z 30, 46, and 76 corresponding to NO, NO2, and N2O3, respectively. A substantial increase in the m/z 30 peak in the presence of deoxy-Hb(FeII), after an initial lag, corresponds to the generation of NO. No change was seen in the
Discussion
The advantages of membrane inlet mass spectrometry have not previously been applied to studies of the generation of NO from nitrite reactivity with hemoglobin. These advantages include a sensitive, direct, and real-time measure of the accumulation in solution of free, unbound NO. Moreover, this technology is quite adaptable to the goals of many in vivo and physiological experiments. The method has the capability of detecting related nitrogen oxides concurrently, if they accumulate sufficiently
Acknowledgments
We thank Dr. Mark T. Gladwin for the program to deconvolute hemoglobin spectra and Dr. Daniel Kim-Shapiro for helpful discussions. This work was supported in part by a grant from the Thomas H. Maren Foundation, from the NIH (GM25154), and from funds made available by the University of Florida.
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