Superoxide Dismutase Targets NO from GSNO to Cysβ93 of Oxyhemoglobin in Concentrated but Not Dilute Solutions of the Protein
Bibliographic record
Abstract
The role of hemoglobin (Hb) in transmitting the vasodilatory property of NO throughout the vascular system is of much current interest. NO exchange between Hb and low-molecular-weight nitrosothiols such as S-nitrosoglutathione (GSNO) has been speculated and reported in vitro. Previously, we reported that NO delivery from GSNO to Cysbeta93 of human oxyHb is prevented in the presence of the Cu chelators, neocuproine, and DTPA.(1) In the present work, 5 mM solutions of commercial human Hb were found by ICP-MS to contain approximately 20 microM Cu and Zn, suggesting the presence of Cu,Zn-superoxide dismutase (CuZnSOD), which was confirmed by Western blotting. SOD activity measurements were consistent with the presence of approximately 20 microM CuZnSOD monomer in 5 mM Hb solutions, which is the physiological concentrations of these proteins in the red blood cell. Incubation of 3.75 mM oxyHb (15 mM heme; 7.5 mM Cysbeta93) with 3.75 or 7.5 mM GSNO gave rise to 50% or 100% S-nitrosation, respectively, of Cysbeta93 as monitored by FTIR nu(SH) absorption, whereas excess GSNO over Cysbeta93 converted oxyHb to metHb due to the reaction, oxyHb + NO<==>metHb + NO(3)(-). Removal of CuZnSOD by anion-exchange chromatography yielded an oxyHb sample that was unreactive toward GSNO, and replacement with bovine CuZnSOD restored reactivity. Addition of 1 microM GSNO (Cysbeta93/GSNO = 1) to solutions diluted 10(4)-fold from physiological concentrations of oxyHb and CuZnSOD resulted largely in metHb formation. Thus, this work reports the following key findings: CuZnSOD is an efficient catalyst of NO transfer between GSNO and Cysbeta93 of oxyHb; metHb is not detected in oxyHb/GSNO incubates containing close to the physiological concentration (5 mM) of Hb and CuZnSOD when the Cysbeta93/GSNO molar ratio is 0.5 to 1.0, but metHb is detected when the total Hb concentration is low micromolar. These results suggest that erythrocyte CuZnSOD may play a critical role in preserving the biological activity of NO by targeting it from GSNO to Cysbeta93 of oxyHb rather than to its oxyheme.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".