Jane H. Park

Advantages of deuterium modification of nitroxide spin labels for biological epr studies

Abstract The spin label, perdeuterio-N-(1-oxy 1-2,2,6,6-tetramenthyl-4-piperidinyl)maleimide (DMSL) was synthesized and its EPR and saturation transfer EPR spectra were compared to those of the hydrogen analogue, HMSL- The labels were studied as freely tumbling entities and also bound to bovine serum albumin (BSA). Significant gains in spectral resolution and detectability were observed for DMSL relative to HMSL.

EPR and saturation transfer EPR studies on glyceraldehyde 3‐phosphate dehydrogenase

Electron paramagnetic resonance (EPR) and saturation transfer–EPR (ST–EPR) techniques were employed to investigate the hydrodynamic properties of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Both apo‐ and holoenzyme were spin‐labeled at the active site cysteine‐149 residue with N‐ (1‐oxyl‐2,2,6,6‐tetramethyl‐4‐piperidinyl) ‐ maleimide. The apo‐ and holoenzymes were observed to have the same hydrodynamic structure and the spectroscopic results were consistent with these complexes behaving as spheres with hydrated radii of 41 A. The environment of the paramagnetic electron was significantly more polar in the spin‐labeled holoenzyme than in the spin‐labeled apoenzyme, suggesting that either ionic residues are positioned closer to the active site in the holoenzyme or that ionic segments of coenzyme nicotinamide adenine dinucleotide (NAD+) itself may interact with the paramagnetic electron of the maleimide spin label. The dependence of the phase quadrature second harmonic absorption ST‐EPR signal upon mic...

The role of the nicotinamide moiety of NAd+ for negative cooperativity in glyceraldehyde-3-phosphate dehydrogenase as studied by spin-labeled cofactors.

Two derivatives of NAD+ spin-labeled at N6 or C-8 of the adenine ring have been shown previously to be active coenzymes of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). When more than two equivalents of either spin-labeled NAD+ are bound to the tetrameric enzyme, spin-spin interaction is observed in the ESR spectra (Deparade, M.P., Glöggler, K. and Trommer, W.E. (1981) Biochim. Biophys, Acta 659, 422-433). After reduction of enzyme-bound NAD+ spin-labeled at C-8 to the corresponding NADH derivative, the additional peaks due to this spin-spin interaction disappear, which implies that the distance between the two radicals increases. It is proposed that the coenzyme slide further towards the active site upon reduction. ADPR spin-labeled at C-8 binds non-cooperatively, exhibiting a dissociation constant of Kd = 33 microM. Even with 3.5 equivalents bound to the enzyme, spin-spin interaction is not observed. AMP spin-labeled at C-8 combines with two sites per monomer, or a total of eight per tetramer. The respective dissociation constants are Kd1 = 30 microM and Kd2 - 2.3 mM. Phosphate competes with AMP bound to the weak site. Spin-spin interaction is not observed. ATP spin-labeled at C-8 is bound about 10-fold tighter than the corresponding AMP derivative. Four equivalents of ATP are bound per tetramer, but it exhibits no spin-spin interactions. It is concluded that the structure of the pyridine moiety of the coenzymes plays a role in orienting the adenine ring and, thus, affects the cooperativity. The N6 derivative of NAD+ also shows spin-spin interaction; however, only data for the C-8 derivatives are shown in detail.

The synthesis of deuterium-substituted, spin-labeled analogues of AMP and NAD+ and their use in ESR studies of lactate dehydrogenase

Two spin-labeled analogues of AMP and NAD+ were synthesized, in which a perdeuterated nitroxide radical (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, TEMPAMINE) was attached to C-6 or C-8 position of the adenine ring. The ESR spectra of these derivatives exhibit a 4-fold increase in sensitivity and a concomitant decrease in line-width as compared to the corresponding protonated analogues. The improved resolution of composite spectra consisting of freely tumbling and immobilized components is demonstrated in ternary complexes of the spin-labeled NAD+ derivatives with lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and oxalate.

Catalytic mechanism and interactions of NAD+ with glyceraldehyde-3-phosphate dehydrogenase: correlation of EPR data and enzymatic studies

Perdeuterated spin label (DSL) analogs of NAD+, with the spin label attached at either the C8 or N6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and conformational changes of the DSL-NAD+-enzyme complex during the catalytic reaction. C8-DSL-NAD+ and N6-DSL-NAD+ showed 80 and 45% of the activity of the native NAD+, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism of this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD+ in three conditions: (1) DSL-NAD+ freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD+ tightly bound to enzyme subunits remote (58 A) from other NAD+ binding sites, and (3) DSL-NAD+ bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 A or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD+ in these three environments and measurements of the binding constants, K1-K4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD+ to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraldehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 A) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD+ complex. When glyceraldehyde-3-phosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the phosphoglyceryl residue and DSL-NAD+. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD+. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD+ from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.

High resolution spin labeled fatty acid: synthesis and EPR spectral characteristics

Abstract Stearic acid spin label substituted with deuterium in all positions and 15 N in the paramagnetic group has been synthesized and was found to display a 5.5 fold gain in sensitivity in the EPR spectrum and a 60% decrease in linewidth compared to the unmodified analog.

Advantages of 15N and Deuterium Spin Probes for Biomedical Electron Paramagnetic Resonance Investigations

15N and deuterium spin labels significantly advanced the application of electron paramagnetic resonance (EPR) in the fields of biomedical science and molecular biology. Problems involving the motional dynamics of enzyme catalysis, protein/ protein interactions, and membrane structure, which heretofore could not be addressed with EPR techniques, can now be investigated with highly quantitative approaches. The remarkable improvements afforded by isotopic substitutions of 15N and deuterium are due to three factors: (1) simplification of EPR spectral line shapes by reduction in the number of peaks; (2) increased sensitivity of the spin labels as evidenced by the elevated amplitude of spectral peak heights; and (3) enhanced resolution caused by narrowing of peak widths and elimination of overlaps of peaks. These factors facilitate the accurate interpretation of EPR spectral data, particularly for structure-function studies of large proteins and membranes. The purpose of this review is to examine the extraordinary utility of the isotopically substituted spin labels by evaluation of selected EPR investigations which may have wide applicability for biomedical experimentation. Therefore, we shall not present a detailed review of recent literature, but rather outline the uses of these spin labels for structure-function studies of enzymes and for the interactions of proteins and membranes in normal and pathological states.

Proton Endor of maleimide spin labeled human hemoglobin

Abstract ENDOR spectra of the spin label N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide bound to lyophilized human oxyhemoglobin suggest a principal element of 3.7 MHz for the largest proton hyperfine tensor. The absence of orientation selection justifies the use of an isotropic broadening factor in the computer simulation of EPR and ST EPR spectra.

Effects of phloretin on lipid organization in the erythrocyte membrane as measured by EPR

Abstract Phloretin is a lipophilic compound which has been widely studied as a broad spectrum effector of metabolite transport in red blood cells (RBC). Phloretin effects on the organization of lipids in the RBC membrane are investigated using the spin-labeled fatty acids, 5 and 16-nitroxyl stearate (5-NS and 16-NS, respectively). Phloretin at different concentrations produced biphasic effects on the lineshape of the EPR response from 16-NS-labeled RBC. The dependence of these changes on the flat cell orientation with respect to the magnetic field suggested that phloretin promoted lipid order at low concentrations (5 to 40 μM) and disorder at high concentrations (40 to 250 μM). The biphasic effects of phloretin occurred at concentrations which parallel its dual actions on metabolite transfer. Phloretin generally inhibits transport (protein-mediated) and stimulates diffusion (lipid-mediated) processes. The spectroscopic effects were best characterized through second-harmonic, in-phase detection. The possible contribution of other factors to the spectroscopic changes is discussed. When RBC were spin labeled with 5-NS, higher concentrations of the probe were required for adequate detection and only monophasic effects of phoretin were observed. The results suggest that membrane lipids are important in phloretin effects on transport and diffusion processes.

Approaches to the Chemical Synthesis of 15N and Deuterium Substituted Spin Labels

With the increasing application of EPR to an expanding field of biomedical problems, the importance of the design and synthesis of spin labels intensifies. The spin label itself can either significantly enhance the productivity of an investigation or constitute the limiting factor. As illustrated in Chapter 11, isotopic substitution of 15N and/or deuterium in the spin probes substantially improves the quality and quantity of information obtained in the areas of enzyme catalysis and membrane characterization. The same design principles apply to 14N,1H and 15N,2H spin labels; however, the syntheses of isotopically substituted compounds present special problems due to the expense of the isotopes and the stringent requirements for isotopic purity in the product. These problems are addressed in detail in Sections 2-4 below. In Chapter 11 and this Appendix, a circumscribed number of isotopically substituted spin labels have been selected to illustrate the potential for dynamic motional analyses. The scope of the presentation is limited in part by the number of papers available in this relatively new area of isotopic substitution and by the authors’ intent to focus on subjects of interest to the biomedical scientist and molecular biologist.