Biomedical subjects
Publications and source records attributed to B Sarkar.
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A comprehensive investigation of the interaction of Zn(II) and Co(II) with the dipeptide glycyl-L-tyrosine has been carried out. The carboxyl, amino, and tyrosyl pKa values, as well as the distribution of solution complexes, have been determined by analytical potentiometry. The amide pKa value was determined by relating the proton magnetic resonance (PMR) titration behavior of the tyrosyl alpha-hydrogen resonance to an H2-acidity function for concentrated solutions of aqueous base. Both metals behave in a qualitatively similar manner, yielding equivalent species as a function of pH. Both metals formed bis-peptide complexes, involving amino and peptide carbonyl coordination near pH = 8, with Zn(II) demonstrating a substantially higher affinity for the ligand. No evidence could be found for direct, metal-promoted phenolic dissociation, although the tyrosyl pKa value was sensitive to metal binding at other loci on the dipeptide molecule. At high pH, both systems ionized two additional protons. In the Co(II) system, these correspond to amide protons. However, it is not entirely clear whether the protons in the Zn(II) system originate from the peptide linkage or metal-bound water molecules.
Studies were undertaken to investigate the Ni(II)-binding properties of human blood serum and to identify the low-molecular-weight Ni(II)-binding constitutents in the serum. Three Ni(II)-binding fractions were obtained when labeled nickel chloride (63NiCl2) was added to the native serum. Of the total Ni(II), 95.7% was associated with albumin, 4.2% was bound to low-molecular-weight components, and a small fraction, usually less than 0.1% was associated with a high-molecular-weight protein that was eluted in the void volume of Sephadex G-150. Amino acids were shown to be responsible for the low-molecular-weight Ni(II)-binding fraction and L-histidine was found to be the main (Ni(II)-binding amino acid in human blood serum. Compared with albumin, L-histidine was shown to possess a greater affinity for Ni(II). Ni(II)-binding to human albumin became evident only when no more L-histidine was available. Since the concentration of albumin is much higher than the concentration of L-histidine in normal serum, most of the added Ni(II) was associated with albumin. The equilibria between Ni(II)-L-histidine and Ni(II)-albumin may facilitate the transport of Ni(ii) between blood and tissues.
The interaction of Hg(II) with human blood serum was studied at physiological pH. Most of the Hg(II) was found to be associated with the proteins, and only a small fraction was associated with the low-molecular-weight substances in serum. Albumin is the major Hg(II)-binding protein (greater than or equal to 90%) in serum. Among the amino acids, L-cysteine has the highest affinity for Hg(II). In dialyzed serum having equimolar concentrations of Hg(II), albumin, and L-cysteine, the amount of Hg(II) found in the supernatant after ultracentrifugation was about 6--7%. There are preferential Hg(II)-binding sites on the albumin molecule. However, no significant change in the circular dichroism spectrum of albumin was detected until at least two equivalents of Hg(II) were present. Hg(II) can mediate the formation of the albumin dimer as well as a ternary complex of the type albumin-Hg(II)-L-cysteine. The latter presumably plays an important role in the transport of Hg(II) between blood and various tissues.
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The peptide Asp-Ala-His-NH-Me was subjected to removal of its N-terminal residue by transamination and scission. Despite the high affinity of the peptide for Cu2+ ions, they catalysed its transamination smoothly. Two main transamination products were found, a complication previously observed with another peptide with an N-terminal aspartic residue, but their scission gave a single product, Ala-His-NH-Me. This was subjected to a further cycle of transamination and scission, and gave a single product after each step. For scission of transaminated peptides it proved unnecessary to remove them from transamination reagents provided that transamination was stopped with EDTA before adding the scission reagent.
A derivative of the native-sequence tripeptide of the specific Cu(II)-transport site of human serum albumin, L-aspartyl-L-alanyl-L-histidine N-methylamide, was synthesized, and its binding to Cu(II) was examined to determine the influence of the side-chain groups on the Cu(II) binding. The equilibria involved in the Cu(II)-L-aspartyl-L-alanyl-L-histidine N-methylamide system were investigated by analytical potentiometry. Three complex species were found in the pH range 4-10. The same species were identified in both the visible and circular-dichroism spectra. The main species present in the physiological pH range is shown to have the same ligands around the square-planar Cu(II) ion as those reported for albumin and tripeptides diglycyl-L-histidine and its N-methylamide derivative. The results obtained from competition experiments showed that this tripeptide has a higher affinity towards Cu(II) than has albumin itself. The overall findings are compared with those from albumin. At neutral pH the side chains do not play any important role in the Cu(II) binding, but at low pH the beta-carboxyl group of the N-terminal aspartic residue becomes important. A possible competition site on albumin for Cu(II) at low pH is discussed.
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Mixed aquo-N-methylimidazole complexes of Co(II) have been studied as a function of pH to gain a fuller understanding of the metal-binding site in Co(II)-carbonic anhydrase. The inherent affinity of N-methylimidazole for Co(II) has been calculated along with a species distribution for the stepwise addition of ligand to the metal ion. From these studies, it is apparent that the occurrence of Zn(II) rather than Co(II) in native carbonic anhydrase can be explained by the stronger affinity of Zn(II) for imidazole and the preference of Zn(II) for a tetrahedral geometry as offered by the enzyme. Octahedral Co(II) fails to ionize metal bound water. However, at high pH, Co(II)-N-methylimidazole complexes interact directly with the hydroxide ion, generating species with visible spectra very similar to that of Co(II)-carbonic anhydrase. Tentative structures have been proposed for these species.
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Six chelating drugs were administered to rats by im injection at equimolar dosages in order to compare their relative effectiveness in prevention of death after a single parenteral injection of NiCl2. Triethylenetetramine and d-penicillamine were most effective antidotes for acute Ni (II)-toxicity. In order of decreasing antidotal effectiveness, diglycyl-L-histidine-N-methylamide, sodium diethyldithiocarbamate and calcium disodium versenate significantly reduced the acute mortality of rats following ip injection of Ni (II). Alpha-Lipoic acid was not effective as an antidote for acute Ni (II)-toxicity.
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The catalytic activity of carbonic anhydrase (EC 4.2.1.1) is linked to the ionization of a group in close proximity to the essential zinc ion. Studies have been undertaken to delineate the ionizations germane to the active-site chelate system. Several imidazole ligand systems were studied in order to approach a representative chelate. The simplest involved the complexation of Zn(II) by imidazole and by N-methylimidazole. As well, two bidentate systems, Zn(II)-4,4'-bis-imidazoylmethane and Co(II)-cyclic-L-histidyl-L-histidine were investigated. It was found that in a species containing metal-bound water and imidazole coordinated by means of the pyridinium nitrogen, the most acidic group was the pyrrole N-H in the imidazole ring. By the use of N-methylimidazole, the pK(a) of a metal-bound water molecule in a tri-imidazole ligand field was found to be 9.1. Noting the preference for labilization of the pyrrole hydrogen, the catalytic features of carbonic anhydrase are reexamined assuming that the pK(enz) is associated with the N-H ionization, and not with the ionization of metal-bound water.
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