Abstract
Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as an in vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 +/- 17 nm in human arterial erythrocytes and 69 +/- 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the beta-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery.
Highlights
Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as an in vivo reservoir for Nitric oxide (NO) from which NO release is allosterically linked to oxygen release
We have developed methodologies to selectively oxidize the NO first from iron-nitrosyl-hemoglobin followed by reduction of the S-NO bond from hemoglobin in solutions of I3Ϫ, releasing NO gas from the cysteine for ozone-based chemiluminescent detection [4, 5, 8], whereas other laboratories first cleave the S-NO linkage with mercury and measure the NO levels using ultraviolet light photolysis [12, 16]
Presence of millimolar glutathione [16, 18, 19, 21], and 3) the difficulty of distinguishing NO derived from two distinct sites on hemoglobin, the heme group and -cysteine 93
Summary
Vol 277, No 31, Issue of August 2, pp. 27818 –27828, 2002 Printed in U.S.A. S-Nitrosohemoglobin Is Unstable in the Reductive Erythrocyte Environment and Lacks O2/NO-linked Allosteric Function*. There have been a number of challenges to the second core principle of the SNO-Hb hypothesis, that NO is released during the oxygen-linked conformational shift of hemoglobin from its R- to T-state Both kinetic and thermodynamic arguments have been made to support the allosterically mediated release of NO from SNO-Hb [12, 14, 16], the physiological relevance of this possible linkage has been challenged on the basis of the very high oxygen affinity of SNO-Hb, potentially limiting its role in basal regulation of NO/oxygen delivery (18 – 20), and the oxygen-independent kinetics of the reaction of. It remains possible that SNO-Hb may participate in NO-dependent events in vivo during pathological conditions associated with red cell oxidation or under circumstances where NO generation is increased in response to infection or pharmacological NO treatment
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