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A new analytical hyphenated technique is proposed for determination and characterization of thiolic proteins, based on reverse phase chromatography (RPC) coupled on-line with cold vapour generation atomic fluorescence spectrometry (CVGAFS). Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0moll-1 urea and p-hydroxymercurybenzoate (PHMB). The derivatized proteins are separated on a C4 Vydac Reverse Phase column. Post-column on-line reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of both the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury, also in the presence of methanol in the RPC eluent phase. Hg(II) is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg°. Under optimized conditions, on-line bromine treatment gives a 98 ± 2% recovery of both free and protein-complexed PHMB. The effect of methanol on the sensitivity of Hg(II) detection was studied and controlled. RPC-CVGAFS system has been applied to the analysis of metallothioneins from rabbit liver (MTRL) standard solutions, and their commercial isoforms MT-1 and MT-2. The analysis of denatured, PHMB-complexed MTs allowed the determination of the number of thiolic groups complexed by PHMB. It was found that MTs from rabbit liver have 10.0 ± 0.3 (MT-1) and 6.7 ± 0.3 (MT-2 and MTRL) –SH groups complexed by PHMB. The detection limit (LODc) for PHMB in 95% methanol in the optimized conditions was about 9.3 × 10-9moll-1 and for the denatured MTs LODc was about 8.6 × 10-10moll-1, taking into account an approximate complexating ratio PHMB:MTs of 7:1. [Copyright &y& Elsevier]

Hydrophobic-interaction chromatography coupled on-line with chemical-vapor-generation atomic-fluorescence spectrometry (HIC-CVGAFS), optimized recently for the analysis of thiol-containing proteins under denaturing conditions, has been used to study the chemical reduction of denatured proteins. Four proteins chosen as models (human serum albumin (HSA), bovine serum albumin (BSA),a-lactalbumin (a-Lac) from bovine milk, and lysozyme from chicken egg (Lys)) were denatured with urea and reduced with dithiothreitol (DTT), with selenol as catalyst. The method is based on derivatization of the -SH groups of proteins withp-hydroxymercurybenzoate (PHMB), followed by HIC separation and post-column on-line reaction of the derivatized reduced, denatured proteins with bromine generated in situ. HgII, derived from rapid conversion of uncomplexed and protein-complexed PHMB, is selectively detected by AFS in an Ar/H2 miniaturized flame after sodium borohydride (NaBH4) reduction to Hg°. The yield of the reduction was studied as a function of reductant concentration, reduction time (tred), and urea concentration. Results showed that the optimum values for DTT and selenol concentrations and fortred were between 1 and 100 mmol L-1 and between 1 and 20 min, respectively, depending on the protein studied. The percentage disulfide bond reduction increases as the urea concentration used for protein denaturation increases, giving a single-step sigmoid increment for single-domain, low-MW proteins (a-Lac and Lys), and a two-step sigmoid increment for multi-domain, high MW proteins (HSA and BSA). The shapes of plots of percentage reduced disulfide against urea concentration are characteristic of each protein and are correlated with the location of S-S in the protein. Under the adopted conditions complete protein denaturation is the conditio sine qua non for obtaining 100% S-S reduction. The detection limit for denatured, reduced proteins examined under the optimized conditions was found to be in the range 1-5×10-12 mol L-1 (10-30 pg), depending on the protein considered. [ABSTRACT FROM AUTHOR]

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC–CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol dm−3 urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0 M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg2+, present in solution as Hg2+–urea complex, is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100±2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and β-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC–CVGAFS apparatus depends on number of &z.sbnd;SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10−10–10−12 mol dm−3, depending on the considered protein, with linear calibration curves spanning over four decades of concentration. [Copyright &y& Elsevier]

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1., (Copyright © 2013 Elsevier B.V. All rights reserved.)