Search PubMed⌕ Search

Biomedical subjects

B Sarkar

Publications and source records attributed to B Sarkar.

At least 91 records · Page 5Linked to original sources
PubMed1985

Peptide and carbohydrate complexes of nickel in human kidney.

The predominant renal and urinary forms of nickel consist of low-Mr complexes. Similarities in the nature of these complexes have been found in kidneys of rats exposed parenterally to NiCl2 and in rat kidneys treated with NiCl2 in vitro. Similar complexes have also been identified after treatment of bovine and human renal soluble fractions with NiCl2. The bulk of nickel in all cases is associated with sulphated oligosaccharide fractions containing uronic acids and neutral sugars. This binding is non-specific, and nickel is readily displaced from these fractions by copper. Smaller amounts of nickel are bound to an acidic peptide, which was purified from human kidneys and partially characterized. Nickel was not displaced from this material by copper at physiological pH. These nickel complexes have not been found in plasma, suggesting that ligand exchange occurs during or after glomerular filtration of the metal.

Amino Acids↗
PubMed1985

Direct evidence of nitrogen coupling in the copper(II) complex of bovine serum albumin by S-band electron spin resonance technique.

ESR spectra of the tight binding Cu(II) complex of bovine serum albumin (BSA) has been studied using S-band. At physiological pH, only one form of copper binding to BSA was detected from the ESR spectra. From previous X-band ESR spectra, nitrogen superhyperfine splittings were observable in the g perpendicular region; however, the resolution of the g parallel region was not sufficient to confirm the exact donor atoms of the complex. Using low-frequency ESR (2-4 GHz) at 77 K, we have resolved the nitrogen superhyperfine structure in the g parallel region. A computer simulation method has been developed for distinguishing between three and four nitrogen donor atoms. The Hyde-Froncisz theory of g and A strain broadening has been modified to use a field-swept calculation for the line shape. The observed intensity pattern and the computer simulation of such spectra positively confirm the structure of Cu(II) ion coordinated to four in-plane nitrogen atoms in frozen aqueous solutions of Cu(II)-BSA complexes at physiological pH. This is the first time that this binding site has been confirmed on the protein instead of a protein fragment or model compound. This work is another example of the usefulness of the S-band ESR technique for characterizing the metal-protein interactions when random variation in g factors cause line broadening in conventional X-band ESR spectra.

Animals↗
PubMed1985

Isolation, purification and 13C- and 1H-n.m.r. assignments of peptide [1-24] of human serum albumin.

Isolation, purification and 360 MHz 1H- and 13C-n.m.r. spectra of the residue corresponding to the NH2-terminal peptide fragment [1-24] of human serum albumin are reported. The various resonances have been assigned to individual amino acid residues and their spatial microenvironment has been determined in a straightforward manner on the basis of (i) pH dependent chemical shifts; (ii) combined use of multiple and selective proton-decoupled 1H- and 13C-n.m.r. spectra; (iii) the characteristic pK values exhibited by protons adjacent to sites of ionization in the molecule; and (iv) comparison of the spectra with the NH2-terminal tripeptide segment of human albumin. The pK values of different ionizable groups all fall in the normal range expected for each titrating sites and support a model of peptide fragment [1-24] in which there is no special structure-forming strong associations. These results are in agreement with those obtained by CD spectroscopy.

Amino Acid Sequence↗
PubMed1984

Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by 13C and 1H NMR spectroscopy.

As a basis for understanding the role of albumin in the transport of metal ions, detailed investigations have been carried out to elucidate the structure of Ni(II)- and Cu(II)-binding site of the peptide residue corresponding to the NH2-terminal peptide fragment 1-24 of human serum albumin by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous medium at different pH values and at different ligand/metal ratios. The results show the following: (i) Diamagnetic Ni(II) complex and paramagnetic Cu(II) complex are in slow exchange NMR time scale. (ii) Titration results of Ni(II)-bound form of peptide 1-24 show the presence of a 1:1 complex in the wide pH range (6.0-11.0), and the same stoichiometry is proposed for Cu(II) as well. (iii) Analysis of the spectra suggests that both Ni(II) and Cu(II) have one specific binding site at the NH2-terminal tripeptide segment (Asp-Ala-His...) involving the Asp alpha-NH2, His N(1) imidazole, two deprotonated peptide nitrogens (Ala NH and His NH), and the Asp COO- group. (iv) Complexation of Ni(II) and Cu(II) causes conformational change near the metal-binding site of the polypeptide chain, but there is no other binding group involved besides those in the first three residues.

Binding Sites↗
PubMed1984

Studies of copper(II) binding to glycylglycyl-L-tyrosine-N-methyl amide, a peptide mimicking the NH2-terminal copper(II)-binding site of dog serum albumin by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy.

Unlike human serum albumin (HSA), dog serum albumin (DSA) does not possess the characteristics of the specific first binding site for Cu(II). In DSA, the important histidine residue in the third position, responsible for the Cu(II)-binding specificity in HSA, is replaced by a tyrosine residue. In order to study the influence of the tyrosine residue in the third position of DSA, a simple model of the NH2-terminal native sequence tripeptide of DSA, glycylglycyl-L-tyrosine-N-methylamide (GGTNMA) was synthesized and its Cu(II)-binding properties studied by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy. The species analysis indicated the existence of five mono-complexes at different protonation states: MHA, MA, MH-1A, MH-2A, MH-3A, and only one bis-complex MH-2A-2. The complexing ability of GGTNMA to Cu(II) was found to be weaker than that of the Cu(II) binding peptide models of HSA. The visible absorption spectra of Cu(II)-GGTNMA complexes are similar to those observed in the case of DSA-Cu(II) complexes. The weaker binding and the spectral properties of Cu(II)-GGTNMA complexes are consistent with less specific Cu(II)-binding properties of the peptide of this sequence similar to what was noted with DSA. CD results are in excellent agreement with species analysis and visible spectra where it is clearly evident that Cu(II) binds to GGTNMA starting from the alpha-NH2 group and step by step to deprotonated amide nitrogens as the pH is raised. The absence of any charge transfer band around 400 nm strongly indicates that Cu(II) does not bind to the phenolate group. Furthermore, NMR results are consistent with the noninvolvement of the tyrosine residue of GGTNMA in Cu(II) complexation. Thus, it is clear that the low Cu(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the NH2-terminal region of the protein.

Animals↗
PubMed1984

Comparative studies of manganese(II)-, nickel(II)-, zinc(II)-, copper(II)-, cadmium(II)-, and iron(III)-binding components in human cord and adult sera.

The binding of six trace metals, Mn(II), Ni(II), Zn(II), Cu(II), Cd(II) and Fe(III), to human cord serum has been studied by Sephadex G-100 gel filtration at physiological pH, using radioisotopes as tracers. The results are compared with those obtained from adult serum. In both cord and adult sera, extensive amounts of the metals are bound to high molecular weight proteins. Among them, Fe(III) is mostly bound to transferrin; Ni(II), Zn(II), Cu(II), and Cd(II) are bound to albumin and other macromolecules. The binding of Mn(II) either to transferrin or albumin is not resolved. Small fractions of Zn(II), Cu(II), and Cd(II) and large fractions of Mn(II) and Ni(II) are found to be associated with low molecular weight components of both sera. The distribution varies from metal to metal. However, the low molecular weight component of the size 1500 - 10000 is present in all the metals studied. Further purification of this component was attempted by DEAE-cellulose ion-exchange chromatography. The possible identity as well as the biological role played by this particular component of serum in the transport of metals in blood and across membranes is discussed.

Cadmium↗
PubMed1983

N.m.r. and e.p.r. investigation of the interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth-modulating tripeptide from plasma.

Interaction of Cu(II) and Gly-His-Lys, a growth-modulating tripeptide from plasma, was investigated by 13C- and 1H-n.m.r. and e.p.r. spectroscopy. The n.m.r. line-broadening was interpreted in terms of major and minor species formed as a function of pH. The results indicate that the n.m.r. line-broadening is due to the presence of minor species in rapid exchange and not due to the major species in solution, which has a large tau M. It is concluded that the technique of 13C- and 1H-n.m.r. line broadening, caused by paramagnetic Cu(II) ion, should be undertaken with caution, since the method may not be useful for obtaining structural information on the major species. The e.p.r. spectra over a wide pH range are almost entirely due to similarly co-ordinating species. Starting at pH 5.5, the narrowest absorption near 340 mT shows superhyperfine structure, which comes out sharply in the pH region 6.0-9.6. The spectra in this pH range showed the seven lines of nitrogen superhyperfine splitting, indicating clearly the co-ordination of three nitrogen atoms to Cu(II). The e.p.r. parameters in the medium pH range, A parallel = 19.5 mT and g parallel = 2.21, fit well with the contention that Cu(II) is ligated to Gly-His-Lys through one oxygen atom and three nitrogen atoms in a square-planar configuration.

Chemical Phenomena↗
PubMed1983

Nickel(II) binding to glycylglycyl-L-tyrosine-N-methyl amide, a peptide mimicking the NH2-terminal nickel(II)-binding site of dog serum albumin: a 1H- and 13C-nuclear magnetic resonance investigation.

The nonspecificity of dog serum albumin (DSA) for Ni(II) is mimicked by the simplest tripeptide, glycylglycyl-L-tyrosine-N-methyl amide, which forms a planar complex at high pH. In this study, the 1H and 13C nuclear magnetic resonance (nmr) spectra of the free and complexed peptide are reported. As the pH is increased for the free peptide, the deprotonation of the terminal amino group (pKa = 7.94) is reflected most strongly by the chemical shift changes of the NH2-terminal -CH2CO- unit. Large upfield and downfield shifts for the tyrosine C xi, C epsilon and C gamma carbon resonances occur on the ionization of the phenolic hydroxyl group. The planar Ni(II) complex is in slow exchange on the nmr time scale and is of 1:1 stoichiometry. The greater chemical shift changes on Ni(II) coordination are observed from the protons nearest the peptide and amino nitrogens:amide CH3 (-0.704), Tyr(3) alpha-CH (-0.667), Gly(1) alpha-CH2 (-0.382), and Gly(2) alpha-CH2 (-0.519, -0.487). In the 13C spectrum, the Gly(1) C alpha (+7.58) is most affected. The Ni(II) ion is therefore at the center of four coordinating nitrogens. Changes in the coupling constants for the Tyr(3) -CH-CH2- moiety suggests a mainly gauche conformation with the tyrosyl ring positioned above the plane of coordination and a weak bonding interaction with the Ni(II) ion is indicated. These results provide structural information regarding the reduced affinity of DSA for Ni(II).

Animals↗
PubMed1983

Nickel-sequestering renal glycoprotein.

Kidney is the target organ where nickel is accumulated and subsequently excreted in the urine after an intraperitoneal administration of 63NiCl2 in rats. The radioactive nickel is found mostly bound to a low molecular weight protein in kidney, which was isolated, purified, and partially characterized in this study. Homogeneity of this protein was determined by polyacrylamide gel electrophoresis. Amino acid analysis showed the presence of high amounts of glycine and proline and low amounts of phenylalanine, tyrosine, hydroxyproline, and hydroxylysine. The protein was found to be a glycoprotein with a carbohydrate content of 10% (wt/wt). Preliminary carbohydrate analysis showed that this glycoprotein is a high mannose-type containing mannose, galactose/glucose, and glucosamine. On the basis of the amino acid and carbohydrate analyses, the molecular weight of the glycoprotein is about 15,000-16,000. In vitro addition of nickel to the kidney cytosol also showed the presence of this protein. The protein appeared not to be affected or altered in its nickel-binding capacity by intraperitoneal or intravenous administration of actinomycin D, indicating it to be a noninducible protein. The glycoprotein demonstrated many characteristics of renal basement membrane. It is proposed that this protein is either a part of the renal basement membrane or is a part of the procollagen in the process of its conversion to collagen of the renal basement protein. The protein has a high affinity for nickel. It also may possess a similar binding affinity for other metals as well and may constitute a natural process of handling toxic levels of metals to be excreted.

Amino Acids↗
PubMed1982

Nickel(II) transport in human blood serum. Studies of nickel(II) binding to human albumin and to native-sequence peptide, and ternary-complex formation with L-histidine.

Detailed studies are reported on the Ni(II)-binding site of human serum albumin (HSA) and the results are compared with those obtained from the N-terminal native-sequence peptide, l-aspartyl-l-alanyl-l-histidine N-methylamide (Asp-Ala-His-NHMe). Equilibrium dialysis of HSA and Ni(II) in 0.1m-N-ethylmorpholine/HCl buffer, pH 7.53, demonstrates a specific Ni(II)-binding site on the protein. l-Histidine, the low-molecular-weight Ni(II)-binding constituent of human serum, is shown to have a greater affinity for Ni(II) than does HSA. A small but significant amount of ternary complex HSA-Ni(II)-l-histidine is also present in the equilibrium mixture containing the three components. The log (association constant) values for the binary and ternary Ni(II) complexes are 9.57 and 16.23 respectively. The complex equilibria between Asp-Ala-His-NHMe and Ni(II) have been investigated by analytical potentiometry in aqueous solution (0.15m-NaCl, 25 degrees C). Several species, including MA, MA(2), MH(-2)A, and MH(-1)A(2) [where M and A represent Ni(II) ion and anionic peptide respectively], were detected in the system, MH(-2)A being the major complex species. Equilibrium studies involving Asp-Ala-His-NHMe, Ni(II) and l-histidine reveal the presence of a ternary complex MH(-1)AB (where B represents anionic l-histidine) at physiological pH. Detailed studies of visible-absorption spectra of HSA in the presence of Cu(II) and Ni(II) reveal that the two metal ions bind HSA at the same site. The visible-absorption spectrum of Ni(II)-HSA complex shows a highly absorbing peak at 420nm (epsilon(max.) = 137; with shoulder at 450-480nm) characteristic of a square planar or square pyramidal co-ordination arrangement about the metal ion. Similar visible-absorption characteristics were observed for the major species MH(-2)A in the Asp-Ala-His-NHMe-Ni(II) system (lambda(max.) = 420nm; epsilon(max.) = 135; with shoulder at 450-480nm). The combination of experimental results from the protein studies and the peptide analyses provides strong evidence for the structure of the Ni(II)-binding site of HSA as one that involves the alpha-amino nitrogen atom, two deprotonated peptide nitrogen atoms, the imidazole nitrogen atom and the side-chain carboxy group of the aspartic acid residue. On the basis of the results obtained from the individual ternary systems involving protein and peptide, a mechanism for the transportation of Ni(II) in the serum is proposed.

Binding Sites↗
PubMed1982

The non-specificity of dog serum albumin and the N-terminal model peptide glycylglycyl-L-tyrosine N-methylamide for nickel is due to the lack of histidine in the third position.

Equilibrium dialysis of dog serum albumin (DSA) against Ni(II) in 0.1 M-N-ethylmorpholine/HCl, pH 7.53, demonstrates the absence of a specific Ni(II)-binding site in DSA. To evaluate at the molecular level the influence of the genetic substitution of L-tyrosine for L-histidine at the N-terminal of DSA, a simple model tripeptide of the N-terminal residues, glycylglycyl-L-tyrosine N-methylamide, was synthesized and its Ni(II)-binding properties studied. A comparison of the visible absorption characteristics of Ni(II)-DSA with those of Ni(II)-glycylglycyl-L-tyrosine N-methylamide reveals a similar change from octahedral to planar co-ordination as the pH is increased. Both systems exhibit a low Ni(II)-binding affinity at physiological pH, with DSA binding a greater percentage of Ni(II) owing to the availability of at least two binding sites of similar affinities. The complex equilibria between Ni(II) and glycylglycyl-L-tyrosine N-methylamide were studied by analytical potentiometry (0.15 M-NaCl, 25 degrees C). Four major complex species, MHA, MH-1A2, MH-2A2 and MH-3A [where M and A represent Ni(II) ion and anionic peptide respectively], were detected, MHA being the single species at physiological pH. There is no evidence for the involvement of the phenolic hydroxy group in the octahedral MHA complex, or within the plane of co-ordination in the high-pH species. The results provide direct evidence that the low Ni(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the N-terminal region of the protein.

Animals↗
PubMed1981

The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth-modulating tripeptide from plasma.

The interaction between Cu(II) and the growth-modulating tripeptide glycyl-L-histidyl-L-lysine in the presence and absence of L-histidine was investigated by potentiometric titration and visible-absorption spectrophotometry at 25 degrees C in 0.15 M-NaCl. Analyses of the results in the pH range 3.5--10.6 indicated the presence of multiple species in solution in the binary system and extensive amounts of the ternary complexes in the ternary system. The species distribution and the stability constants, as well as the visible-absorption spectra of the species, were evaluated. The combined results were used to propose the structure of some of the complexes. The influence of the epsilon-amino group of the peptide in the enhancement of the stability constants was reflected prominently when compared with those complexes formed by either glycyl-L-histidine or glycyl-L-histidylglycine. The results obtained from the equilibrium-dialysis experiments showed that this tripeptide was able to compete with albumin for Cu(II) at pH 7.5 and 6 degrees C. At equimolar concentrations of albumin and the peptide, about 42% of the Cu(II) was bound to the peptide. At the physiologically relevant concentrations of Cu(II), albumin, L-histidine and this peptide, about 6% of the Cu(II) was associated with the low-molecular-weight components. This distribution could be due to the binary as well as the ternary complexes. The possible physiological role of these complexes in the transportation of Cu(II) from blood to tissues is discussed.

Copper↗
PubMed1981

Electron spin resonance study of the copper(II) complexes of human and dog serum albumins abd some peptide analogs.

Electron spin resonance spectra of the first Cu(II) complexes of human serum albumin, dog serum albumin, L-aspartyl-L-histidine N-methylamide and glycyl-gly-cyl-L-histidine N-methylamide have been studied using isotopically pure 65Cu in its chloride form. At 77 degrees K, the esr spectra of Cu(II) complex of human serum albumin exhibited only one form of esr signal between pH 6.5 and 11. No intermediate forms were detected. The presence of an equally spaced nine-line superhyperfine structure with spacing approximately 15 G indicated considerable covalent bonding between Cu(II) and four nitrogen atoms derived from the protein. The esr spectrum form of Cu(II) bound to human serum albumin detected at neutral pH would be consistent with the participation of four nitrogens from the alpha-NH2 group, two peptide groups, and the imidazole group of a histidine residue. In contrast, the esr spectra of Vu(II)-dog serum albumin complex showed a transition from a low pH form to a high pH form as the pH was increased to 9.5. These spectral changes were found to be reversible upon lowering the pH. Ligand superhyperfine splitting in the low pH form of the esr signal of Cu(II)-dog albumin were not resolved. The distinct pH dependence of the esr signals observed in human and dog serum albumin complexes could be correlated to their respective optical spectra changes as a function of pH. At room temperature and in the pH range between 6 and 11, the esr spectra of Cu(II) complexes of L-aspartyl-L-alanyl-L-histidine N-methylamide and glycyl-glycyl-L-histidine N-methylamide exhibited a well-resolved nine-line superhyperfine structure indicating metal coordination with four equivalent nitrogen atoms of peptide.

Animals↗
PubMed1981

Molecular design of a cyclic heptapeptide to mimic the zinc-binding site of carbonic anhydrase. Synthesis and zinc-binding studies by 13C-and 1H-N.M.R. spectroscopy.

A cyclic heptapeptide [cyclo-(Gly-L-His-Gly-L-His-L-Gly-L-Gly)] was designed to mimic the Zn(II)-binding site of carbonic anhydrase. The cyclic heptapeptide was synthesized from the linear heptapeptide, Gly-L-His-Gly-L-His-Gly-L-His-Gly-OH, which in turn was obtained by coupling of the fragments, viz. BOC-Gly-L-His-Gly-N3 and L-His-GLy-L-His-Gly-OBzlNO2 followed by deblocking of amino and carboxyl protecting groups. Conversion of the linear heptapeptide to the azide by treatment with diphenylphosphoryl azide was followed by cyclization in high dilution. A homogeneous material was isolated by counter-current distribution followed by gel filtration. It was found to be ninhydrin negative. The n.m.r. spectrum of the material upon integration indicated the proper ratios of various kinds of protons to be expected of the cyclic heptapeptide. A detailed 13C- and 1H-n.m.r. investigation was undertaken to determine the Zn(II)-binding ligands of the cyclic heptapeptide. The assignments for all the resonances were attempted by spin-decoupling method, pH and solvent effects, and by comparison of resonances of similar protons and carbons of model peptides. The n.m.r. titration results of the Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)-binding, the changes in the chemical shift of the imidazole protons were relatively large, indicating that this ring is involved in the complexation. All the peptide -NH-resonances were observable and unaffected; consequently, none of these nitrogens can serve as a ligand. In the case of 13C resonances, addition of 1 equiv. of Zn(II) to the cyclic heptapeptide, te C(2), C(4), and C(5) carbon resonances of this group were dramatically affected and showed a very large change in chemical shift upon complexation. The results demonstrate that Zn(II) binds to all three imidazole residues of the designed cyclic heptapeptide.

Binding Sites↗