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Updated: Aug 22, 2026
Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication
Published on: September 20, 2016
Structural investigation of pig metmyoglobin by 129Xe NMR spectroscopy
Marcella Corda1, Benedetta Era, Antonella Fais
1Dipartimento di Scienze Applicate ai Biosistemi, Cittadella Universitaria di Monserrato, 09042 Monserrato, CA, Italy.
Abstract:
The potentiality of xenon's sensitivity to its local magnetic environment is thoroughly investigated to probe internal structural differences between pig and horse metmyoglobin (MMb). These MMb's differ by 14 amino acids. One of these, Ile142 in horse MMb, is located in the proximal cavity, which is the xenon-binding site in horse MMb, and is replaced by Met142 in pig MMb. Specific and non-specific xenon-protein interactions are investigated here by 129Xe NMR chemical shifts and relaxation rate in aqueous solutions of pig MMb as a function of the xenon and protein concentrations. The results are complemented with 129Xe NMR data of the isostructural carbonmonoxy myoglobin (COMb), with computational calculations in order to highlight the structural differences between the cavities, and 1H NMR spectra to test the dependence of the 1H chemical shift on the addition of xenon. The 129Xe chemical shift NMR parameters are analysed quantitatively in terms of a two-site model. Xenon forms a 1:1 complex with the protein, characterized by an equilibrium binding constant K=[Xe]in/([Xe]out[MMb]), and exchanges rapidly between a cavity within the protein (X(ein)) and all other environments (Xe(out)). A comparison of equilibrium constant, K (74 M(-1)) in pig and K (146 M(-1)) in horse, reveals differences in affinity of xenon to the interior of pig MMb. Changes in xenon binding in both pig and horse MMb are also pointed out by other experimental results, e.g. the difference in the estimated delta(in), which is shifted downfield in pig MMb and upfield in horse MMb, with respect to 129Xe in buffer solution; the xenon-iron distance, 7.4 A, which is longer in the pig than was found in the horse, 5.3 A.
Insights
Xenon-129 NMR reveals structural differences between pig and horse metmyoglobin (MMb). Horse MMb shows higher xenon binding affinity than pig MMb due to distinct amino acid compositions in the xenon-binding cavity.
Area of Science:
- Biophysics
- Structural Biology
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- Metmyoglobin (MMb) from different species, like pigs and horses, exhibits structural variations due to differing amino acid sequences.
- These structural differences, particularly in the xenon-binding cavity, can influence the interaction of small molecules like xenon with the protein.
Purpose of the Study:
- To investigate the potential of xenon's sensitivity to its magnetic environment for probing internal structural differences between pig and horse metmyoglobin (MMb).
- To characterize specific and non-specific xenon-protein interactions in MMb using 129Xe NMR.
Main Methods:
- Utilized 129Xe NMR chemical shifts and relaxation rates in aqueous solutions of pig and horse MMb.
- Analyzed data using a two-site exchange model to determine equilibrium binding constants and xenon-binding site characteristics.
- Complemented NMR data with computational calculations and 1H NMR spectra.
Main Results:
- Quantified differences in xenon binding affinity, with horse MMb exhibiting a higher equilibrium binding constant (146 M⁻¹) compared to pig MMb (74 M⁻¹).
- Observed distinct 129Xe chemical shift parameters (delta(in)) for pig (downfield shift) and horse (upfield shift) MMb relative to buffer solution.
- Determined differences in xenon-iron distances: 7.4 Å in pig MMb versus 5.3 Å in horse MMb.
Conclusions:
- Xenon-129 NMR is a sensitive tool for detecting subtle structural variations within the MMb cavities between species.
- The amino acid substitution at position 142 (Ile in horse, Met in pig) significantly impacts xenon's binding affinity and interaction dynamics within the MMb protein.
