Use of the vanadyl(IV) ion as a new spectroscopic probe of metal binding to proteins.  Vanadyl insulin

Chasteen, N. Dennis; DeKoch, Robert J.; Rogers, B L; Hanna, Melvin W.

doi:10.1021/ja00785a048

Cited by 73 publications

(44 citation statements)

References 5 publications

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“…As a control, bovine serum albumin which contains -SH group(s) gave a pronounced yellow color indicative of the presence of a sulfhydryl group. The rotational correlation time for this site, as estimated from line widths (Chasteen and Hanna, 1972), is -5 X 1O-Io sec as compared with = l o m 8 sec expected for the enzyme as a whole. This suggests that we may be observing monodentate coordination to the enzyme.…”

Section: Structure Ofthe a (Active) Sitementioning

confidence: 59%

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Kinetics and electron paramagnetic resonance spectra of vanadyl(IV) carboxypeptidase A

Rj¹,

Dj²,

Jc³

et al. 1974

Biochemistry

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This study was undertaken to further investigate the use of the vanadyl ion, V02+, as a spectroscopic probe of active sites in metalloenzymes. The vanadyl derivative of zinc bovine carboxypeptidase A was prepared and found to hydrolyze the ester hippuryl-L-P-phenyllactic acid and the peptide benzoylglycyl-L-phenylalanine. One vanadyl ion per enzyme molecule was sufficient for full activity. Electron paramagnetic resonance (epr) spectra of polycrystalline samples were examined and three types of binding sites, A, B, and C, were found. The A site corresponds to the active site normally occupied by zinc and does not appear unusual in its geometry about the V 0 2 + group. Two water molecules and two imidazole groups, His-69 and His-196, appear to bind in equatorial positions while the axial w e have been investigating the use of the vanadyl ion, V 0 2 + , as a spectroscopic probe of metal binding sites in proteins. In addition to its infrared and visible spectroscopic properties, the vanadyl ion exhibits sharp room temperature and liquid nitrogen electron paramagnetic resonance (epr) spectra which are sensitive to the ligand environment about the V 0 2 + group (Chasteen et al., 1973;Boucher et al.,

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Section: Structure Ofthe a (Active) Sitementioning

confidence: 59%

“…Fitzgerald and Chasteen, 1974b. X-Ray results on carbonic anhydrase have been wmmariied by Lindskog et af., 1971Chasteen et al, 1973. Blundell er al , 1971.…”

Section: Resultsmentioning

confidence: 99%

Kinetics and electron paramagnetic resonance spectra of vanadyl(IV) carboxypeptidase A

Rj¹,

Dj²,

Jc³

et al. 1974

Biochemistry

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“…In this study we characterized the metal-binding properties of normal NCt (fraction A) by EPR and electron nuclear double resonance (ENDOR) using the vanadyl ion (VO2+) as a paramagnetic probe. The VO2+ ion has been shown to be an effective paramagnetic substitute for many divalent metal ions in metalloproteins and metalloenzymes (7)(8)(9)(10) and is, in particular, an effective substitute for Ca2+ and Mg2+ (11,12). VO2+ is an ideal substitute for Ca2+ because VO2+, like Ca2+, has a high affinity for binding to oxygen-donor ligands (7).…”

mentioning

confidence: 99%

Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Mustafi

Nakagawa²

1994

Proc. Natl. Acad. Sci. U.S.A.

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This inhibitor protein has been isolated in four isoforms from urine of normal subjects (1) and kidney stone patients (2), from surgically removed calcium oxalate kidney stones (3), and from kidney tissue of nine species of vertebrates (4). Among these nine species, the dissociation constant of these inhibitor proteins for binding COM crystals varies from 6 x 10-6 to 4 x 10-8 M. Purification of NC from bovine kidney tissue by DEAE-cellulose chromatography showed that it also consists offour isoforms, which have been designated as fractions A-D (4-6). Fractions A-D of NC were also obtained similarly from other species, as described earlier (1-4). Fractions A and B, the "strong" inhibitors, are abundant in normal subjects (1), whereas stone formers excrete less of fractions A and B and more of C and D, which are "weak" inhibitors (2). NC is an acidic glycoprotein containing 33% acidic amino acid residues (Asp and Glu) and about 5% aromatic and basic amino acid residues (5). Fractions A-D differ according to the content of carbohydrate and phosphate. Also, the strong inhibitors contain three or four y-tcarboxyglutamic acid residues per molecule, which are not present in NC of stone-forming patients (5).We have previously shown that, on the basis of 31P NMR studies of Ca2+-NC complexes and by equilibrium dialysis with 45Ca, a total of 4 mol of Ca2+ are bound per mol of NC (6). In this study we characterized the metal-binding properties of normal NCt (fraction A) by EPR and electron nuclear double resonance (ENDOR) using the vanadyl ion (VO2+) as a paramagnetic probe. The VO2+ ion has been shown to be an effective paramagnetic substitute for many divalent metal ions in metalloproteins and metalloenzymes (7-10) and is, in particular, an effective substitute for Ca2+ and Mg2+ (11,12). VO2+ is an ideal substitute for Ca2+ because VO2+, like Ca2+, has a high affinity for binding to oxygen-donor ligands (7). By EPR spectrometric titration, we have shown that VO2+ binds to NC-A with a stoichiometry of 4 mol/mol of protein. Furthermore, we have also demonstrated that VO2+ competes with Ca2+ in binding to NC. An unusual result of this preliminary study of VO2+-NC complexes obtained by ENDOR spectroscopy showed that the coordinating ligands to the metal ions are from protein residues with complete exclusion of solvent water from the inner coordination sphere. EXPERIMENTAL PROCEDURES Materials. NC was isolated from fresh bovine kidney tissue and separated into four fractions by DEAE-cellulose chromatography (Whatman DE-52), as described (1). These fractions were eluted by varying the ionic strength: NC-A at 10-15 inS, NC-B at 16-23 mS, NC-C at 24-27 mS, and NC-D at 32-38 inS, using a linear gradient ofNaCl from 0.1 M to 0.5 M in 0.05 M Tris HCl at pH 7.3. Fraction A (NC-At), which exhibits the highest affinity for Ca2+ binding, was used in this study. This fraction of protein has been found in normal (non-kidney stone) subjects and can be used as a model system for a strong inhibitor protein. Fraction A of NC was first eluted...

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“…85 In the hexameric 'resting' form of insulin, the subunits are tied together by Zn 2+ ions in an octahedral environment with three facially arranged histidines and water molecules. EPR patterns of the vanadyl ions have been employed since the early 1970s by Chasteen et al to extract specific information on metal-binding site(s) of peptides and proteins such as insulin and carboxypeptidase-A.…”

Section: Nonfunctional Binding Of Vanadate and Vanadyl To Proteinsmentioning

confidence: 99%

Influence of Vanadium Compounds on Cellular Functions

Rehder

2008

Bioinorganic Vanadium Chemistry

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Use of the vanadyl(IV) ion as a new spectroscopic probe of metal binding to proteins. Vanadyl insulin

Cited by 73 publications

References 5 publications

Kinetics and electron paramagnetic resonance spectra of vanadyl(IV) carboxypeptidase A

Kinetics and electron paramagnetic resonance spectra of vanadyl(IV) carboxypeptidase A

Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Influence of Vanadium Compounds on Cellular Functions

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