J. Am. Chem. zyxwvut SOC. zyxwvu 1990, 112, zyxwvu 2901-2908 2901 Application of Time-Resolved 51V2D NMR for Quantitation of Kinetic Exchange Pathways between Vanadate Monomer, Dimer, Tetramer, and Pentamer? Debbie C. Crans,* Christopher D. Rithner,* and Lisa A. Theisen Contribution from the Department zyxwvu of Chemistry, Colorado State University, Fort Collins, Colorado 80523. Received July 3, 1989 Abstract: A two-dimensional "V homonuclear NMR exchange experiment (2D-EXSY) has zyxw been used to study the oligomerization reactions vanadate undergoes in aqueous solutions. This manuscript describes the first quantitative measurement of complex intermolecular chemical exchange rates by using 51V (I zyxwvutsrq = 7/2) in a 2D-EXSY experiment. Microscopic (pseudo-first-order) rate constants for intermolecular exchange were obtained by using a numerical procedure to solve the 2D exchange matrix. The 2D exchange matrix was converted to a rate matrix that could be used in a kinetic analysis of the four exchanging vanadium species. The major pathway for monomer formation is unimolecular decomposition of the dimer. The major pathway for dimer formation is dimerization of the monomer. The tetramer forms mainly from two monomers and one dimer. At low vanadate concentrations, the pentamer forms from tetramer and either monomer or dimer with similar rate. At higher vanadate concentration, the pentamer exchanges more rapidly with the tetramer. The vanadate monomer is involved in more significant reaction pathways than any other species. The vanadate dimer is inherently more labile than the tetramer and pentamer as illustrated by its rapid hydrolysis rate. Our analysis demonstrates an approach that is applicable zyxw to solving other multiexchange systems. The 2D-EXSY method is versatile and may become central to determining the major reaction pathways by which vanadium acts in both chemical and biological systems. Vanadium is an important trace element that has potent bio- logical effects in mammals.' Although vanadium is beneficial at low levels, it can be toxic at higher levels. Despite this, very little characterization of the toxic and beneficial vanadium species have been carried out.' Vanadate is a potent inhibitor for ribonuclease, phosphatases, ATPases, and myosin. The inhibiting species for these enzymes is usually monomeric vanadate, and its activity is presumably due to the stable trigonal-bipyramidal structure that mimics the transition-state structure in the phosphate ester hy- drolysis reactions.' Vanadate polyanions also act as inhibitors. The dimer inhibits phosphoglycerate mutase,& acid phosphatase from seminal fluid,Zb and glucose-6-phosphase dehydrogenase,2c the tetramer inhibits 6-phosphogluconate dehydrogenaseZd and glucose-6-phosphate dehydrogenase,2c and the decamer inhibits Ca2+ ATPase,2e muscle phosphorylase,2f adenylate kinase,2g Aqueous solutions of vanadate anions contain several oligomers including significant concentrations of monomer, dimer, tetramer, and pentamer. The s'V NMR spectra can be used to determine the speciation of vanadate oligomers. The eqs 1-3 describe the and phosphofructokinase.2g 2v, zyxwvutsr 2 v, relationships observed in aqueous s ol~tions.~~~~~~-~ The structures of the vanadate derivatives in solution are somewhat controver- siaL3p4 The tetramer and pentamer are generally believed to be c~clic~,~ although the cyclic structure for the tetramer has recently been questioned.6 Numerous additional minor species have been observed in 51V N M R spectra and were assigned according to their s'V N M R chemical shifts.* The equilibrium position is affected by pH, ionic strength, temperature, and vanadate concentrations. The rates and mechanisms of conversion between various anions are not well understood. Since we believe that the reactions between vanadate species are important in understanding the chemical and biochemical properties of vanadate, we have used a quantitative s'V N M R homonuclear exchange experiment (2D-EXSY) to directly determine exchange rates and rate con- stants between the vanadate species. 'Dedicated to the late Professor John K. Stille. Experimental Section General Methods. Chemicals were reagent grade (Fisher, Aldrich) and used without further purification. Water was distilled and deionized. A vanadate stock solution was prepared by dissolving vanadium pentoxide with 2 equiv of sodium hydroxide to generate a vanadate solution of 0.25 M; this solution was stored at 4 OC. The concentrations of the vanadate standard solutions were monitored by UV spectroscopy (at wavelengths from 260 to 270 nm), and no changes in concentrations were observed over the course of 6 months. 1D ST NMR Spe~troscopy.~-~ s'V NMR spectra were recorded at 52.6 MHz on a Bruker WPSY spectrometer (4.7 T) (Figure I), 79 MHz on a Bruker ACP-300 NMR spectrometer (7.0 T), and 132 MHz on a Bruker AM-500 spectrometer (1 1.7 T). For the ID spectra we typically use spectral windows of 150 ppm, a 90° pulse angle, and an aquisition time of about 0.1 s with no relaxation delay between pulses.5b An ex- ponential line broadening of 15 Hz was applied to the FID prior to Fourier transformation. The chemical shifts are reported relative to the external reference standard, V0Cl3 (0 ppm), although we in practice use an external reference solution (pH 7.5) containing the complex of va- nadate and diethanolamine (-490 p~m).~* The DEA complex is a con- venient reference because the s%' NMR resonance varies only slightly with pH and ionic strength, and the signal appears in the chemical shift range of interest. The 1D spectral integration, using the mole fraction for each resonance combined with the known total vanadium concen- tration, allowed calculation of the concentrations of various vanadate oligomers. The T,'s were obtained by using a Freeman-Hill modified inversion recovery experiment. The Tl's were obtained from the slope of semilog (1) (a) Nechay, B. R.; Nanninga, L. B.; Nechay, P. S. E.; Post, R. L.; Grantham, J. J.; Macara, I. G.; Kubena, L. F.; Phillips, T. D.; Nielsen, F. H. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1986,45, 123-32. (b) Chasteen, N. D. Struct. Bonding 1983, 53, 105-38. (2) (a) Stankiewicz, P. J.; Gresser, M. J.; Tracey, A. S.; Hass, L. F. Biochemistry 1987,26, 1264-9. (b) Crans, D. C.; Simone, C. M.; Saha, A. K.; Clew, R. H. Biochem. Biophys. Res. Commun. 1989, 165, 246-50. (c) Crans, D. C.; Schelble, S. Biochemistry. In press. (d) Crans, D. C.; Willging, E. M.; Butler, S. R. J. Am. Chem. SOC. 1990, 112, 427-32. (e) Csermely, P.; Martonosi, A.; Levy, G. C.; Ejchart, A. J. Biochem. J. 1985, 230, 807-15. (f) Soman, G.; Chang, Y. C.; Graves, D. J. Biochemistry 1983,22,4994-5ooO. (9) Boyd, D. W.; Kustin, K.; Niva, M. Biochem. Biophys. Acta 1985, 827, 472-5. (3) Pope, M. T. Heteropoly and Isopoly Oxometalates; Springer-Verlag: New York, 1983. (4) (a) Heath, E.; Howarth, 0. W. J. Chem. Soc., Dalton Trans. 1981, 1105-10. (b) Howarth, 0. In Multinuclear NMR; Mason, J., Ed.; Plenum: New York. 1988; Chapter 5. (5) (a) Gresser, M. J.; Tracey, A. S. J. Am. Chem. Soc. 1985, 107, 4215-20. (b) Crans, D. C.;Shin, P. K. Inorg. Chem. 1988,27, 1797-1806. (c) Crans, D. C.; Bunch, R. L.; Theisen, L. A. J. Am. Chem. Soc. 1989, Ill, (6) Tracey, A. S.; Gresser, M. J.; Galeffi, B. Inorg. Chem. 1988, 27, 7597-601. 157-61. 0002-7863/90/ 151 2-2901$02.50/0 0 1990 American Chemical Society