A direct spectrophotometric method for the simultaneous determination of zinc and cobalt in metalloproteins using 4-(2-pyridylazo)resorcinol

Säbel, Crystal E.; Shepherd, Jeffrey L.; Siemann, Stefan

doi:10.1016/j.ab.2009.05.007

Cited by 40 publications

(41 citation statements)

References 13 publications

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“…− , which can readily react with bulky organic compounds [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] to give ternary complexes with good extraction behavior and analytical potential. In the present paper, we investigated the complex formation in a liquidliquid extraction system containing Co(II), PAR, and Nitron (Nt).…”

Section: Introductionmentioning

confidence: 99%

Complex Formation in a Liquid-Liquid Extraction System Containing Cobalt(II), 4-(2-Pyridylazo)resorcinol, and Nitron

Racheva

Gavazov

Lekova

et al. 2013

Journal of Materials

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Complex formation and liquid-liquid extraction were studied in a system containing cobalt(II), 4-(2-pyridylazo)resorcinol (PAR), 1,4-diphenyl-3-(phenylamino)-1H-1,2,4-triazole (Nitron, Nt), water, and chloroform. The effect of some experimental parameters (pH, shaking time, concentration of PAR, and concentration of Nt) was systematically investigated, and the optimum conditions for cobalt extraction as an ion-association complex, (NtH + )[Co 3+ (PAR) 2 ], were found. The following key equilibrium constants were calculated: constant of association (Log = 4.77 ± 0.06), constant of distribution (Log D = 1.34 ± 0.01), and constant of extraction (Log ex = 6.11 ± 0.07). Beer's law was obeyed for Co concentrations up to 1.7 g mL −1 with a molar absorptivity of 6.0 × 10 4 L mol −1 cm −1 at max = 520 nm. Some additional characteristics, such as limit of detection, limit of quantification, and Sandell's sensitivity, were estimated as well.

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Section: Introductionmentioning

confidence: 99%

Complex Formation in a Liquid-Liquid Extraction System Containing Cobalt(II), 4-(2-Pyridylazo)resorcinol, and Nitron

Racheva

Gavazov

Lekova

et al. 2013

Journal of Materials

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“…In experiments described as "metal free," enzyme preparations were treated with chelating resin to remove exogenous metals (as described for the ICP-MS assays), and all buffers were constructed with HPLC grade water. Prior to beginning kinetic measurements, the buffers were assayed for residual zinc, which was found to be approximately 300 nM by the PAR absorbance assay (23,48). For all substrates tested, enzyme and substrate were preincubated separately at 30°C for 5 min prior to mixing.…”

Section: Methodsmentioning

confidence: 99%

“…IMP-1 enzymes purified as described above were dialyzed overnight against 150 mM NaCl in 100 mM Tris-HCl at pH 7.0 containing a packet of Chelex resin, followed by addition of 50% glycerol by volume for storage at Ϫ20°C. To confirm that one zinc ion remained per enzyme, proteins were tested with the PAR assay as described above (see Table S2 in the supplemental material) (23,48). The kinetic profile of the C221G IMP-1 mutant was determined in metal-free 50 mM HEPES buffer containing 20 g/ml BSA at pH 7.0, with 10 or more nitrocefin dilutions with concentrations between 3 and 100 M. The C221G IMP-1 enzyme was stored on ice and incubated in the buffer for 5 min at 30°Ϯ 2°C prior to the addition of the nitrocefin.…”

Section: Methodsmentioning

confidence: 99%

Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

Banks

Shanker

Mikulski

et al. 2012

Antimicrob Agents Chemother

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e Metallo-␤-lactamases catalyze the hydrolysis of a broad range of ␤-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ␤-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ␤-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ␤-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 M. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.A n increasing prevalence of antibiotic-resistant strains is reducing the available options for treating bacterial infections. ␤-Lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobial agents (31). The major contributors to ␤-lactam antibiotic resistance are ␤-lactamase enzymes, which act by hydrolyzing the four-member ␤-lactam ring (15,62). Mechanistically, this is accomplished either via an active-site serine in the class A, C, and D enzymes or through the use of one or two Zn 2ϩ ions (class B) (4). Class B metallo-␤-lactamases (M␤Ls) are a group of structurally similar enzymes that exhibit a characteristic ␣␤/␤␣ sandwich fold, with the active site located at the interface between domains. This scaffold supports up to 6 residues at the active site that coordinate either one or two zinc ions that are central to the catalytic mechanism (1,6,35). M␤Ls have the capacity to hydrolyze most clinically available ␤-lactam drugs, including extended-spectrum cephalosporins and carbapenems (8,10,32,40,59,61).The IMP-1 metallo-␤-lactamase has been identified in several nosocomial, Gram-negative, pathogenic bacteria, including Pseudomonas aeruginosa and Serratia marcesens (22, 28). The bla IMP-1 gene is carried on an integron element that facilitates genetic transfer and is likely why IMP-1 is found among multiple bacterial species (28, 58). The combination of a broad ␤-lactam antibiotic substrate profile and the genetic ca...

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“…To assess whether cobalt was the cation present in the catalytic center, we carried out (i) spectrophotometric complexation with 4-(2-pyridylazo)resorcinol (50) and (ii) inductively coupled plasma mass spectrometry (ICP-MS) on liquid protein samples treated with aqua regia (10% [vol/vol]). ICP-MS was also carried out on dissolved crystals.…”

Section: Methodsmentioning

confidence: 99%

“…This result is not surprising, as no divalent metal was included during the crystallization experiments and a lack or half-occupancy of cations in this conserved bimetallic center has been observed in analog structures (e.g., PDB IDs 1VGY, 1XMB, 2F7V, 2FH8, 2IMO, 2POK, 2RB7, 3CT9, and 3GB0). Cobalt was chosen as the metal in the BsLcar structure, based on the recorded spectra of protein samples treated with 4-(2-pyridylazo)resorcinol (50). Liquid BsLcar samples (0.06 mol monomeric species) were also analyzed by ICP-MS, giving 301 Ϯ 3 ppb of Co 2ϩ (0.05 mol), resulting in a ratio of 0.8 atom of Co 2ϩ per monomer of BsLcar.…”

Section: R369 H219(a) N260(a)mentioning

confidence: 99%

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

et al. 2012

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c N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg 234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family. N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases; EC 3.5.1.87) irreversibly hydrolyze the amide bond of the carbamoyl group in L-N-carbamoyl-amino acids. Due to their stereospecificity, L-carbamoylases are widely used in kinetic resolution for the production of optically pure amino acids (see reference 44 and references therein). Furthermore, their substrate promiscuity allows their use in different multienzymatic processes, increasing their economic and industrial relevance (48). L-Carbamoylases have been isolated and characterized from different sources, although most of the studies carried out have focused on biotechnological applications (44). Their relationship with other amidohydrolases and with the peptidase M20/M25/M40 family has been established based on sequence similarity (21,29,42). A closer relationship among L-carbamoylases and ␤-ureidopropionases is supported by the findings of two ␤-ureidopropionases that are able to hydrolyze N-carbamoyl-L-␣-amino acids (40,41,47).In a previous work, we were able to show the involvement in catalysis of several highly conserved residues in L-carbamoylases, as well as the relationship of these enzymes with several peptidases (42). Dimerization was also proved to be necessary for enzymatic activity, as the residues involved in catalysis come from both monomers. In this work, we determined the first crystal structure of an L-carbamoylase, belonging to Geobacillus stearothermophilus CECT43 (BsLcar). Mutagenesis studies of BsLcar confirmed the importance of conserved residues in this enzyme. Unexpectedly, a highly conserved arginine in L-carbamoylases has been proved critical for the dimerization of the enzyme, and thus for enzymatic activity. Comparative studies revealed striking characteristics of the different "dimerization" domains of the peptidase M20/M25/ M40 family, in agreement with previous evolutionary hypotheses on this family of enzymes (3, 37) and suggesting new evolutionary considerations. Finally, an alternative re...

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A direct spectrophotometric method for the simultaneous determination of zinc and cobalt in metalloproteins using 4-(2-pyridylazo)resorcinol

Cited by 40 publications

References 13 publications

Complex Formation in a Liquid-Liquid Extraction System Containing Cobalt(II), 4-(2-Pyridylazo)resorcinol, and Nitron

Complex Formation in a Liquid-Liquid Extraction System Containing Cobalt(II), 4-(2-Pyridylazo)resorcinol, and Nitron

Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

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