Molecular design to mimic the copper(II) transport site of human albumin: studies of equilibria between copper(II) and glycylglycyl-L-histidine-N-methyl amide and comparison with human albumin

1976 ◽  
Vol 54 (8) ◽  
pp. 1300-1308 ◽  
Author(s):  
Theo P. A. Kruck ◽  
Show-Jy Lau ◽  
Bibudhendra Sarkar
Keyword(s):  
Ternary System ◽  
Equilibrium Dialysis ◽  
Human Albumin ◽  
Binary Complex ◽  
Mixed Complex ◽  
Free Peptide ◽  
System L ◽  
Peptide Derivative ◽  
Carboxyl Terminal ◽  
Transport Site

In continuing the investigation of designing the specific Cu(II)-transport site of human serum albumin, the peptide derivative glycylglycyl-L-histidine-N-methyl amide was designed to approximate more closely to the native protein. This peptide derivative was synthesized in good yield. The equilibria involved in the binary system, Cu(II)–glycylglycyl-L-histidine-N-methyl amide, have been studied, as well as those in the ternary system, L-histidine–Cu(II)–glycylglycyl-L-histidine-N-methyl amide. This peptide derivative was found to bind Cu(II) exclusively as a 1:1 complex in the pH range 4 to 11, having the same ligand atoms as those for the carboxyl-terminal free peptide and human albumin. However, it was found that glycylglycyl-L-histidine-N-methyl amide bound Cu(II) more strongly than did glycylglycyl-L-histidine, the stability constants being log β1–21 = −0.479 and −1.99 respectively. In the ternary system, only 10% of the mixed complex was detected at pH 7, in comparison to 80% found in the case of the carboxyl-terminal free peptide. This finding agrees well with the increased stability of this peptide binary complex. These observations are also consistent with the results obtained from the equilibrium dialysis experiments. The Cu(II) – peptide amide complex has a dissociation constant of 2.07 × 10−17, indicating a higher binding strength of this peptide derivative for Cu(II) over the native albumin by a factor of 3.

Equilibrium and spectroscopic studies of the ternary system: L-histidine, copper(II), and the native sequence peptide representing the copper(II)-transport site of human serum albumin

1985 ◽  
Vol 63 (11) ◽  
pp. 3117-3121 ◽  
Author(s):  
Masaaki Tabata ◽  
Bibudhendra Sarkar
Keyword(s):  
Human Serum Albumin ◽  
Ternary System ◽  
Serum Albumin ◽  
Human Serum ◽  
Spectroscopic Studies ◽  
Mixed Ligand ◽  
Anionic Form ◽  
System L ◽  
Native Sequence ◽  
Transport Site

Equilibrium and spectroscopic studies of Cu(II)-transfer of native sequence tripeptide, L-aspartyl-L-alanyl-L-histidine-N-methyl amide (AAHNMA), representing the Cu(II)-transport site of human serum albumin (HSA), and L-histidine (L-His) are reported. The equilibria in the ternary system, M–A–B (M = Cu(II), A = anionic form of AAHNMA, and B = anionic form of L-His) have been investigated by analytical potentiometry in I = 0.2 [(Na+,H+) (Cl−,OH−)] at 25 °C. The ternary system shows the presence of five mixed ligand complexes: MH2AB, MHAB, MAB, MH−1AB, and MH−2AB. The species distribution and their stability constants were evaluated by the mathematical analysis of the potentiometric data. The species were further confirmed by their individual spectra computed from the absorption measurements. At physiological pH, the equilibrium studies reveal the presence of 13% of MH−1AB (λmax = 530 nm.ε = 90 M−1 cm−1) and 3% MAB (λmax = 595 nm, ε = 97 M−1 cm−1). The combined results of equilibrium and spectroscopic studies indicate the mixed ligand complex CuH−1AB formed by deprotonation of peptide nitrogen as an important intermediate in the Cu(II)-transfer reaction. The stability constant of CuH−1AB is compared to those of other tripeptides which were designed to mimic the specific Cu(II)-transport site of human albumin.

scholarly journals The kinetic mechanism catalysed by homogeneous rat liver 3α-hydroxysteroid dehydrogenase. Evidence for binary and ternary dead-end complexes containing non-steroidal anti-inflammatory drugs

1991 ◽  
Vol 278 (3) ◽  
pp. 835-841 ◽  
Author(s):  
L J Askonas ◽  
J W Ricigliano ◽  
T M Penning
Keyword(s):  
Rat Liver ◽  
Initial Velocity ◽  
Equilibrium Dialysis ◽  
Hydroxysteroid Dehydrogenase ◽  
Pyridine Nucleotide ◽  
Free Enzyme ◽  
Binary Complex ◽  
Dead End ◽  
Inflammatory Drugs ◽  
Inhibition Studies

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E-NADH complex, and by the failure to detect the binding of either [3H]androsterone or [3H]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [14C]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (Kd = 1-2 microM for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (Kd = 22 microM for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-3H]NADH or pro-S-[4-3H]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-3H]NADH was used, confirming that purified 3 alpha-HSD is a Class A dehydrogenase.

Binding of Al by protein in plasma of patients on maintenance hemodialysis.

1985 ◽  
Vol 31 (8) ◽  
pp. 1314-1316 ◽  
Author(s):  
M Cochran ◽  
D Patterson ◽  
S Neoh ◽  
B Stevens ◽  
R Mazzachi
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Human Albumin ◽  
Hemodialysis Patients ◽  
Single Peak ◽  
Molar Ratio ◽  
Maintenance Hemodialysis ◽  
Mass Fractions

Abstract Gel filtration of plasma from hemodialysis patients, with use of reagents and apparatus with carefully minimized background Al concentrations, reproducibly showed a single peak for Al, corresponding exactly to the elution position of transferrin. The Al/transferrin molar ratio in adjacent fractions was constant (mean 0.126, SE 0.006) in replicate experiments. In contrast, the association of Al with albumin varied. Using both equilibrium dialysis and gel-filtration techniques, in the presence and absence of calcium or phosphate, we could demonstrate no significant binding of Al by human albumin at Al concentrations of 1 to 12 mumol/L. We saw no Al peak in pooled, concentrated, low-molecular-mass fractions of plasma gel-filtered on Sephadex G-50. Evidently, transferrin is the sole Al-binding protein in plasma of hemodialysis patients.

Molecular design to mimic the copper(II) transport site of human albumin. The crystal and molecular structure of copper(II) – glycylglycyl-L-histidine-N-methyl amide monoaquo complex

1976 ◽  
Vol 54 (8) ◽  
pp. 1309-1316 ◽  
Author(s):  
Norman Camerman ◽  
Arthur Camerman ◽  
Bibudhendra Sarkar
Keyword(s):  
Molecular Structure ◽  
Molecular Design ◽  
Crystal And Molecular Structure ◽  
Human Albumin ◽  
Water Molecules ◽  
Copper Binding ◽  
Amino Terminal ◽  
Square Planar ◽  
Cell Dimensions ◽  
Transport Site

Glycylglycyl-L-histidine-N-methyl amide is a copper-binding tripeptide designed and synthesized to mimic the copper-transport site of human albumin. Reddish-purple crystals of the copper-tripeptide amide complex (Cu–GGHa), grown at physiological pH, are triclinic, with cell dimensions a = 9.990, b = 9.986, c = 7.682 Å, α = 107.40, β = 91.72, γ = 96.49°, space group P1, Z = two units of Cu–GGHa and two water molecules per cell. The structure was solved by interpretation of a Cu–phased Fourier map containing a great deal of false symmetry, after multiple attempts with direct phasing methods failed. Refinement proceeded to R = 0.036. The conformations of the two Cu–GGHa units are virtually identical. Each copper is tetradentate chelated by the amino terminal nitrogen, the next two peptide nitrogens, and a histidyl nitrogen of a single tripeptide molecule in a mildly distorted square planar arrangement. The Cu…N distances range between 1.90–2.05 Å, with N…Cu…N angles of 165 and 176°. An oxygen atom provides a fifth position weaker interaction in each case, with Cu…O distances of 2.61 and 2.88 Å.

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