NMR Spectroscopic Approach Reveals Metabolic Diversity of Human Blood Plasma Associated with Protein–Drug Interaction

Although blood plasma has been used to diagnose diseases and to evaluate physiological conditions, it is not easy to establish a global normal concentration range for the targeting components in the plasma due to the inherent metabolic diversity. We show here that NMR spectroscopy coupled with principal component analysis (PCA) may provide a useful method for quantitatively characterizing the metabolic diversity of human blood plasma. We analyzed 70 human blood plasma samples with and without addition of ibuprofen. By defining the PC score values as diversity index (<i>I</i>_div) and the drug-induced PC score value change as interaction index (<i>I</i>_dist), we find that the two indexes are highly correlated (<i>P</i> < 0.0001). Triglycerides, choline-containing phospholipids, lactate, and pyruvate are associated with both indexes (<i>P</i> < 0.0001), respectively. In addition, a significant amount of lactate and pyruvate are in the NMR “invisible” bound forms and can be replaced by ibuprofen. The diffusion and transverse relaxation time weighted NMR approaches gave rise to a better characterization of the diversity and the interaction than that of the one acquired using NOESYPR1D with 100 ms mixing time. These results might be useful for understanding the blood plasma–drug interaction and personalized therapy

Steady-state kinetics studies on (A) heme destruction and (B) peroxidase activity vs pH change of wild-type cyt <i>c</i> and its Y67H mutant.

<p>Steady-state kinetics studies on (A) heme destruction and (B) peroxidase activity vs pH change of wild-type cyt <i>c</i> and its Y67H mutant.</p

Structure of the heme region of cyt <i>c</i> and the location of Tyr67 in the structure (pdb code: 1YIC), showing the hydrogen bond network formed by amino acids His18, Tyr67, Met80, Asn52 and Thr78.

<p>Structure of the heme region of cyt <i>c</i> and the location of Tyr67 in the structure (pdb code: 1YIC), showing the hydrogen bond network formed by amino acids His18, Tyr67, Met80, Asn52 and Thr78.</p

Structural Basis for Cytochrome <i>c</i> Y67H Mutant to Function as a Peroxidase

<div><p>The catalytic activity of cytochrome <i>c</i> (cyt <i>c</i>) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt <i>c</i> Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe³⁺ ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H₂O₂ by functioning as an acidic catalyst.</p></div

Experimental restraints and structural statistics for cyt <i>c</i> Y67H variant.

a<p>The programs PROCHECK and PROCHECK-NMR were used to check the overall quality of the structure and GLY and Pro are excluded from the Ramachandran analysis.</p>b<p>For the PROCHECK statistic, less than 10 bad contacts per 100 residues, and an overall G-factor larger than −0.5 are expected for a good quality structure.</p><p>Experimental restraints and structural statistics for cyt <i>c</i> Y67H variant.</p

1D ¹H-NMR spectra of cyt <i>c</i>.

<p>(A) The high-field shifted region of the native cyt c (down) and its Y67H variant (upper) spectra. (B) The down-field shifted region of the native cyt <i>c</i> (down) and its Y67H variant (upper) spectra. The peaks in 1D ¹H-NMR spectrum were assigned as (1a) His18 Hε1, (2a) His71 Hε1, (3a) His71 Hδ2, (4a) heme 8-CH₃, (5a) His18 Hδ2, (6a) heme 3-CH₃, (7a) heme 5-CH₃, (8a) His18 Hβ2, (9a) heme 1-CH₃ and (10a) heme 7-Hα2, respectively. The peaks in 1D ¹H-NMR spectrum of native cyt <i>c</i> were assigned as (1b) His18 Hε1, (2b) Met80 ε-CH₃, (3b) Met80 Hγ, (4b) heme 8-CH₃, (5b) heme 3-CH₃, (6b) His18 Hδ2, (7b) heme 7-Hα2, (8b) His18 Hβ2, (9b) heme 7-Hα1 and (10b) His18 Hδ1, respectively.</p

Upon overlaying Cα atoms in second structural region and heme backbone atoms, the conformational comparison: (A) between wild-type cyt <i>c</i> (grey, pdb code 1YIC) and its Y67H variant (green); (B) between the Y67F (<i>magenta</i>, pdb code 1CTY) and Y67H (green) mutants; (C) between Y67H (green) and alkaline form (red, pdb code 1LMS).

<p>Upon overlaying Cα atoms in second structural region and heme backbone atoms, the conformational comparison: (A) between wild-type cyt <i>c</i> (grey, pdb code 1YIC) and its Y67H variant (green); (B) between the Y67F (<i>magenta</i>, pdb code 1CTY) and Y67H (green) mutants; (C) between Y67H (green) and alkaline form (red, pdb code 1LMS).</p

The possible mechanism for guaiacol reaction with H₂O₂ catalyzed by the cyt <i>c</i> Y67H mutant at pH 4.0.

<p>The possible mechanism for guaiacol reaction with H₂O₂ catalyzed by the cyt <i>c</i> Y67H mutant at pH 4.0.</p

New assignments of Y67H mutant.

<p>New assignments of Y67H mutant.</p

Heme destruction of the wild-type cyt <i>c</i> (A) and its Y67H variant (B) as indicated by dissipation of the Soret band.

<p>Spectra were scanned every 20 sec, and the arrows indicate the direction of change. The concentrations of H₂O₂ were 10 mM in (A) and 1 mM in (B), the pH condition in both cases is 6.0.</p