Figure 3.

Biochemical and spectroscopic properties of [6-¹³C,¹⁵N₃]-arginine. (A) Enzyme kinetics of recombinant human arginase-1, using either natural abundance arginine (red) or [6-¹³C,¹⁵N₃]-arginine (blue) as the enzymatic substrate. Data points were fit to the Michaelis–Menten kinetics model (dashed lines) to approximate K_m and V_max, revealing no significant difference in K_m but a significant 13% decrease in V_max with isotopic enrichment. (B) Inversion recovery and (C) CPMG acquisitions on the carbon-6 resonance of each arginine variant at 14.1 T, which were used to measure T₁ and T₂, respectively. At 14.1 T, ¹⁵N-enrichment does not increase carbon-6 T₁ but it yields a roughly 3-fold increase in T₂. (D) 1 T ¹³C NMR spectrum of an aqueous equimolar mixture of [6-¹³C]- and [6-¹³C,¹⁵N₃]-arginine, displaying the carbon-6 resonance of each arginine variant. Peak widths were quantified to approximate relative differences in carbon-6 T₂*, and the narrower peak widths for [6-¹³C,¹⁵N₃]-arginine indicate an increase in T₂* with ¹⁵N-enrichment. (E) 1 T ¹³C NMR of HP [6-¹³C]- and [6-¹³C,¹⁵N₃]-arginine acquired with a 30° excitation every 3 s. Scalar-mediated relaxation from adjacent ¹⁴N-nuclei results in depolarization of the carbon-6 resonance of [6-¹³C]-arginine during sample transfer from the polarizer to the spectrometer, whereas this relaxation mechanism is mitigated in [6-¹³C,¹⁵N₃]-arginine.