Subunit Disassembly and Unfolding Kinetics of Hemoglobin Studied by Time-Resolved Electrospray Mass Spectrometry
Bibliographic record
Abstract
We report the use of electrospray ionization (ESI) mass spectrometry (MS) in conjunction with online rapid mixing to monitor the kinetics of acid-induced ferrihemoglobin denaturation. Under equilibrium conditions, the hemoglobin mass spectrum is dominated by the intact heterotetramer. Dimeric and monomeric species are also observed at lower intensities. In addition, ionic signals corresponding to hexameric (tetramer-dimer) and octameric (tetramer x 2) hemoglobin species are observed. These complexes may represent weak solution-phase assemblies. The acid-induced denaturation process was monitored for reaction time ranging from 9 ms to approximately 3 s. The data obtained were subjected to a global analysis procedure which simultaneously fit all kinetic (ESI-MS intensity vs time) profiles to multiexponential expressions. Results of the global analysis are consistent with the coexistence of two subpopulations of tetrameric hemoglobin which differ in their disassembly rates and ESI charge states. The higher-charge state tetramer ions preferentially dissociate via a rapid pathway (tau(1) = 51 ms), resulting in the transient formation of a heme-saturated dimer, holo-alpha-globin, and a heme-deficient dimer. The latter is shown by MS/MS to be comprised of a heme-bound alpha-subunit complexed with an apo-beta-chain. The slow-decaying tetramer population, apparent at a slightly lower average charge state, breaks down into its monomeric constituents with no observable intermediate species (tau(2) = 390 ms). Surprisingly, unfolded apo-alpha-globin is formed more rapidly than unfolded apo-beta-globin. The appearance of the latter occurs with a relaxation time tau(3) of 1.2 s. It is postulated that accumulation of unfolded apo-beta-globin is delayed by transient population of an undetected unfolding intermediate.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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) | 0.001 | 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 teacher head, 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".