Binding of copper(II) ion to cytochrome c

1981 ◽  
Vol 34 (1) ◽  
pp. 91 ◽  
Author(s):  
MA Augustin ◽  
JK Yandell
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Binding Site ◽  
Spectral Data ◽  
Binding Sites ◽  
Association Constant ◽  
Association Constants ◽  
Visible Absorption ◽  
Ph Range ◽  
Horse Cytochrome

Binding of aquacopper(11) ion to horse and tuna ferricytochrome c has been investigated over the pH range 2-9. Between pH 4 and 7 the principal binding site for horse cytochrome has an intrinsic association constant of 1.8 × 104 1. mol-1 and pKa of 6.5�0.6 at 25�C and ionic strength of 0.1. Binding to histidine 33 is consistent with this information, as well as with e.s.r., n.m.r. and visible absorption spectral data. Other sites also bind copper but with association constants at least a factor of ten smaller. At high pH (> 8) further binding sites predominate.

scholarly journals An H.P.L.C. Study of Cooperative Guest Binding by a Covalently Linked b-Cyclodextrin Dimer

1997 ◽  
Vol 50 (8) ◽  
pp. 857 ◽  
Author(s):  
Anna K. Croft ◽  
Christopher J. Easton ◽  
Stephen F. Lincoln ◽  
Bruce L. May ◽  
John Papageorgiou
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
High Performance ◽  
Association Constant ◽  
Association Constants ◽  
Cooperative Binding ◽  
Cyclodextrin Dimer ◽  
Guest Binding ◽  
Covalently Linked ◽  
Single Binding Site

The complexation of the anions of Methyl Orange (MO¯) (1) and Indomethacin (Ind-) (2) by the β-cyclodextrin dimer (3) has been studied by means of high-performance liquid chromatography. The cyclodextrin annuli are shown to jointly act as a single binding site for complexation of MO¯ (1). Thus the association constants of (3·3±0·3) × 103 and (220±20) × 103 dm3 mol−1 for the 1 : 1 complexes with β-cyclodextrin and the dimer (3), respectively, indicate very strong cooperative binding by the annuli of the linked species. By contrast, the complexation of Ind¯ (2) by the cyclodextrin dimer (3) is characterized by two independent binding sites. This lack of cooperative binding is reflected in the association constant for the 1 : 1 complex in each binding site, which at (1·3±0·1) × 103 dm3 mol−1 is little greater than that of (0·7±0·05) × 103dm3 mol−1 for the complex with β-cyclodextrin.

Characterisation of Metal Binding Sites for 8-Azaguanine and 8-Azahypoxanthine Derivatives Synthesis and X-Ray Structural Analysis of Methylmercury(II) and Zinc(II) Complexes

1986 ◽  
Vol 41 (9) ◽  
pp. 1117-1122 ◽  
Author(s):  
W. S. Sheldrick ◽  
P. Bell
Keyword(s):  
Structural Analysis ◽  
Binding Site ◽  
Metal Binding ◽  
Binding Sites ◽  
Antineoplastic Agent ◽  
X Ray ◽  
Metal Binding Sites ◽  
Ph Values ◽  
Ph Range

Abstract The complexes [(CH3Hg)AGuaH ] (1) and [(CH3Hg)2AGua] • H2O (2) have been isolated from aqueous 1:1 and 2:1 solutions of CH3HgOH and 8 -azaguanine (AGuaH2) at respective pH values of 5 and 9. Only one CH3Hg+ complex of 8 -azahypoxanthine (AHxH2), namely [(CH3Hg)2AHx] (3), could be isolated under analogous conditions. X-ray structural analyses established N1 and N9 as metal binding sites in 3 and N9 as the coordination position in [Zn(H2O)4(AHxH)2] (4). With 8-aza-9-benzylhypoxanthine (9-BzAHxH) only one CH3Hg+ complex [(CH3Hg)9-BzAHx] (5) could be isolated in the pH range 2-10. N1 was established by X-ray structural analysis as the binding site. The relevance o f these findings to an understanding of ligand behaviour of the antineoplastic agent 8 -azaguanine is discussed.

The determination of calcium-binding sites of human erythrocyte membranes

1984 ◽  
Vol 62 (6) ◽  
pp. 398-408 ◽  
Author(s):  
R. Blaine Moore ◽  
E. E. Dryden ◽  
D. I. C. Kells ◽  
J. F. Manery
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Plasma Membranes ◽  
Equilibrium Dialysis ◽  
Association Constants ◽  
Erythrocyte Membranes ◽  
Least Squares Regression ◽  
Calcium Binding Site ◽  
Calcium Binding Sites

Calcium binding to leaky erythrocyte plasma membranes was measured by three different procedures: Millipore filtration, equilibrium dialysis, and partition centrifugation. The curve derived from the binding equation, which best fit the means of the raw data, was used to estimate the association constants and capacities of the binding sites. A computer program (Gaushaus) which uses a nonlinear, least-squares regression protocol was also used to confirm these estimates. On the basis of these analyses we propose the presence of three classes of calcium-binding sites with the following apparent association constants and capacities: site 1, Ka = 3 × 104 M−1 and n = 30 nmol/mg protein; site 2, Ka = 3 × 103 M−1 and n = 200 nmol/mg protein; site 3, Ka = ~102 M−1 and n = ~200 nmol/mg protein. Calcium binding to erythrocyte membranes sealed in a high-salt solution showed the presence of site 3, but not site 2. The influence of phospholipids on the binding of calcium was evaluated by pretreating ghosts with phospholipase C (Clostridium welchii, EC 3.1.4.3). Treatment with this enzyme removed 80% of the total membrane phosphorus, predominantly from sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. By the method of partition centrifugation two classes of binding sites were identified by computer analysis. Their association constants and capacities are, respectively, 1.1 × 105 M−1 and 20 nmol/mg protein for site 1 and 4.4 × 103 M−1 and 200 nmol/mg protein for site 2. We speculate that calcium-binding site 1 is composed of acidic phospholipids, calcium-binding site 2 is composed of spectrin and actin, and calcium-binding site 3 is composed of sialic acid.

scholarly journals Histidine residues of zinc ligands in β-lactamase II

1978 ◽  
Vol 175 (2) ◽  
pp. 441-447 ◽  
Author(s):  
G S Baldwin ◽  
A Galdes ◽  
H A O Hill ◽  
B E Smith ◽  
S G Waley ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Binding Site ◽  
Binding Sites ◽  
Zinc Binding ◽  
Histidine Residue ◽  
Beta Lactamase ◽  
Histidine Residues ◽  
Zinc Enzyme ◽  
Zinc Binding Site ◽  
Ph Range

1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0–9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.

Binding of zinc to bovine and human milk proteins

1989 ◽  
Vol 56 (2) ◽  
pp. 235-248 ◽  
Author(s):  
Harjinder Singh ◽  
Albert Flynn ◽  
Patrick F. Fox
Keyword(s):  
Ionic Strength ◽  
Binding Sites ◽  
Binding Capacity ◽  
Equilibrium Dialysis ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Association Constants ◽  
Zn Concentration ◽  
Β Lactoglobulin ◽  
Scatchard Plots

SummaryZn binding by whole bovine and human casein and by purified bovine caseins and whey proteins was investigated by equilibrium dialysis. Bovine αs1 casein had the greatest Zn-binding capacity (˜ 11 atoms Zn/mol). Protein aggregation was observed as Zn concentration was increased and- the protein precipitated at a free Zn concentration of 1·7 mM. Zn binding increased with increasing pH in the range 5·4–7·0 and decreased with increasing ionic strength. Competition between Zn and Ca was observed for binding to αs1-casein indicating common binding sites for these two metals. Bovine β-casein bound up to 8 atoms Zn/ mol and precipitated at a free Zn concentration of ˜ 2·5 mM, while K-casein bound 1–2 atoms Zn/mol. Whole bovine and human casein bound 5–8 atoms Zn/mol and precipitated at a free Zn concentration of ˜ 2·0 mM. Scatchard plots for Zn binding to caseins showed upward convexity, possibly due to Zn-induced association of caseins. Apparent average association constants (K¯app) for all caseins were similar (log K¯app 3·0–3·2). Enzymic dephosphorylation of αs1- or whole bovine casein markedly reduced, but did not eliminate, Zn binding. Thus, phosphoserine residues appeared to be the primary Zn-binding sites in caseins. With the exception of bovine serum albumin. which bound over 8 atoms Zn/mol, the bovine whey proteins, β-lactoglobulin, α-lactalbumin and lactotransferrin, had little capacity for Zn binding.

scholarly journals A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A

2000 ◽  
Vol 350 (3) ◽  
pp. 933-941 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
Peter TOMME ◽  
Emily A. AMANDORON ◽  
Douglas G. KILBURN
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Streptomyces Lividans ◽  
Association Constants ◽  
Carbohydrate Binding ◽  
Toxin B ◽  
Ricin Toxin ◽  
Functional Sites ◽  
Directed Mutation ◽  
Single Binding Site

The C-terminal module of xylanase 10A from Streptomyces lividans is a family 13 carbohydrate-binding module (CBM13). CBM13 binds mono- and oligo-saccharides with association constants of ≈ 1×102 M-1−1×103 M-1. It appears to be specific only for pyranose sugars. CBM13 binds insoluble and soluble xylan, holocellulose, pachyman, lichenan, arabinogalactan and laminarin. The association constant for binding to soluble xylan is (6.2±0.6)×103/mol of xylan polymer. Site-directed mutation indicates the involvement of three functional sites on CBM13 in binding to soluble xylan. The sites are similar in sequence, and are predicted to have similar structures, to the α, β and γ sites of ricin toxin B-chain, which is also in family 13. The affinity of a single binding site on CBM13 for soluble xylan is only ≈ (0.5±0.1)×103/mol of xylan. The binding of CBM13 to soluble xylan involves additive and co-operative interactions between the three binding sites. This mechanism of binding has not previously been reported for CBMs binding polysaccharides. CBM13 is the first bacterial module from family 13 to be described in detail.

Study of Cytochrome c-DNA Interaction – Evaluation of Binding Sites on the Redox Protein

Nanoscale ◽  
2014 ◽  
Vol 6 (22) ◽  
pp. 13779-13786 ◽  
Author(s):  
Christoph Wettstein ◽  
Ciara Kyne ◽  
Aishling M. Doolan ◽  
Helmuth Möhwald ◽  
Peter B. Crowley ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Complex Formation ◽  
Protein Complex ◽  
Binding Sites ◽  
Ph Value ◽  
Dna Interaction ◽  
Redox Protein ◽  
Protein Complex Formation

The characteristics of cyt c-DNA complexation change from transient to permanent in dependency on the pH value and the ionic strength. Hereby, typical binding sites known from cyt c-protein complex formation are affected by DNA.

scholarly journals Covalent association of C3b with C4b within C5 convertase of the classical complement pathway.

1987 ◽  
Vol 165 (6) ◽  
pp. 1494-1507 ◽  
Author(s):  
Y Takata ◽  
T Kinoshita ◽  
H Kozono ◽  
J Takeda ◽  
E Tanaka ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Association Constant ◽  
Affinity Binding ◽  
Ester Bond ◽  
Complement Pathway ◽  
High Affinity Binding ◽  
Classical Complement Pathway ◽  
High Affinity ◽  
Classical Complement

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

scholarly journals Role of the N-terminal region of phospholipase A2 subunit of β1-bungarotoxin in the toxin-Ca2+ complex-formation

1991 ◽  
Vol 278 (2) ◽  
pp. 481-486 ◽  
Author(s):  
S T Chu ◽  
Y H Chen
Keyword(s):  
Phospholipase A2 ◽  
Metal Ions ◽  
Binding Sites ◽  
Association Constant ◽  
Enzymic Activity ◽  
Association Constants ◽  
High Affinity ◽  
Toxin Molecule ◽  
High Affinity Site

beta 1-Bungarotoxin consists of a phospholipase A2 subunit and a non-phospholipase A2 subunit. Modification of beta 1-bungarotoxin with CNBr resulted in cleavage at Met-6 and Met-8 of its phospholipase A2 subunit. Analysis of the fluorescence data of both the toxin-Ca2+ complex at 300-350 nm and the toxin-Tb3+ complex at 450-650 nm showed the existence of two binding sites for both metal ions on the different domains of the toxin molecule. At pH 7.6 the association constants for the high-affinity and low-affinity sites of the toxin-Ca2+ complex were determined to be 2.79 x 10(3) +/- 0.21 x 10(3) M-1 and 0.47 x 10(3) +/- 0.06 x 10(3) M-1 respectively. For the toxin-Tb3+ complex the association constant for the high-affinity site was 2.95 x 10(3) +/- 0.43 x 10(3) M-1 and that for the low-affinity site was 0.11 x 10(3) +/- 0.03 x 10(3) M-1. Removal of the N-terminal octapeptide of the phospholipase A2 subunit from the toxin molecule caused disintegration of the low-affinity site but did not disrupt the high-affinity site. This might accompany a change in the configuration around His-48 of the phospholipase A2 subunit. Between pH 6 and 8 the binding of metal ions to the high-affinity site increased but that to the low-affinity site did not change with increasing pH. The neurotoxicity and enzymic activity of the toxin were lost on removal of the low-affinity site.

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