scholarly journals The effect of N-bromosuccinimide on the sub-unit structure of avidin and its complexes with biotin

1968 ◽  
Vol 110 (1) ◽  
pp. 59-66 ◽  
Author(s):  
N. M. Green ◽  
M. E. Ross
Keyword(s):  
Binding Sites ◽  
Oxidation Products ◽  
Guanidinium Chloride ◽  
Unit Structure ◽  
Tryptophan Residues ◽  
Biotin Binding ◽  
Tetrameric Complex ◽  
Chloride Concentrations ◽  
Avidin Biotin Complex

1. Each molecule of biotin bound to avidin protected four tryptophan residues from oxidation by N-bromosuccinimide, regardless of the occupancy of neighbouring binding sites in the four-sub-unit avidin molecule. 2. The oxidation products from avidin molecules in which some of the sites were occupied were separated on columns of Sephadex G-100. In the absence of biotin, oxidized avidin broke down into sub-units, which partly aggregated. When some of the sites were occupied by biotin, the only detectable products were completely oxidized avidin (sub-units and large aggregates) and unoxidized avidin–biotin complex (tetramer). Since the biotin-containing sub-units were randomly distributed before oxidation took place, they must have dissociated from the molecules containing oxidized sub-units and then reassociated to form the tetrameric avidin–biotin complex. 3. This reassociation still occurred in 3·5m-guanidinium chloride, which prevents the reassociation of unoccupied sub-units. During their brief existence in this medium, the sub-units of avidin–biotin complex were protected from oxidation by N-bromo-succinimide to the same extent as was the tetrameric complex. 4. It is concluded that sub-units of avidin–biotin complex do not readily lose their biotin, even in 3·5m-guanidinium chloride, and that monomeric biotin–binding species are probably present in solutions of avidin sub-units at guanidinium chloride concentrations between 3·0m and 3·5m.

scholarly journals The dissociation of avidin–biotin complexes by guanidinium chloride

1972 ◽  
Vol 130 (3) ◽  
pp. 707-711 ◽  
Author(s):  
N. M. Green ◽  
E. J. Toms
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Guanidinium Chloride ◽  
A Site ◽  
Avidin Biotin Complex

Avidin molecules in which a fraction of the four binding sites were occupied by biotin did not dissociate completely in 6.4m-guanidinium chloride. Only unoccupied subunits dissociated. The remainder recombined to form the tetrameric avidin–biotin complex. The rate at which unoccupied subunits were unfolded and dissociated was only decreased by one-half in species in which three of the four binding sites were occupied by biotin. These results can be explained by assuming that unfolding of unoccupied subunits followed by dissociation from the tetramer is initiated by penetration of guanidinium ions into the binding site and disorganization of this region of the subunit. When a site is occupied by biotin this pathway is blocked and the subunit does not unfold. Each subunit behaves independently and is not markedly stabilized when neighbouring subunits are occupied.

scholarly journals The properties of subunits of avidin coupled to Sepharose

1973 ◽  
Vol 133 (4) ◽  
pp. 687-698 ◽  
Author(s):  
N. Michael Green ◽  
E. John Toms
Keyword(s):  
Spatial Distribution ◽  
Binding Sites ◽  
Binding Capacity ◽  
Similar Rate ◽  
Guanidinium Chloride ◽  
Temperature Dependent ◽  
Low Concentrations ◽  
Biotin Binding ◽  
Covalently Linked ◽  
Sepharose 4B

Avidin that had been coupled to Sepharose 4B activated with CNBr retained over 90% of its biotin-binding capacity. When low concentrations of CNBr were used about 75% of the protein could be removed from the Sepharose by washing with guanidinium chloride (6 m). The remaining 25%, the covalently bound subunits, had an almost undiminished capacity for biotin but a decreased affinity. Addition of avidin subunits in guanidinium chloride to the coupled subunits followed by dilution or dialysis restored the original biotin-binding capacity and affinity. Three classes of binding sites were present in preparations of the subunits. About 25% were weak (K=5X10−8m), about one third exchanged their biotin in a few minutes (K∼10−10m) and the remainder were indistinguishable from the native tetramer. The last-named exchanged their bound biotin at a similar rate at pH5 and at pH2, they did not lose their biotin in 6 m-guanidinium chloride and they were resistant to tryptic digestion in the absence of biotin. The proportion of these stable sites could be increased to 65% when the subunits coupled to Sepharose were incubated at 37°C. This increase was reversed by guanidinium chloride, which suggested that it was caused by a temperature-dependent association of covalently linked subunits. This in turn implies a temperature-dependent mobility of the agarose matrix of the Sepharose. Analysis of the spatial distribution of subunits within the Sepharose beads led to the conclusion that the association of subunits implied that they could move through distances greater than 20nm (several hundred Å). This mobility and consequent formation of tetramer was greatly decreased when avidin subunits were coupled to Sepharose that had been cross-linked with divinyl sulphone.

Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors

1980 ◽  
Vol 58 (12) ◽  
pp. 1323-1334 ◽  
Author(s):  
Nicholas E. Dixon ◽  
John A. Hinds ◽  
Ann K. Fihelly ◽  
Carlo Gazzola ◽  
Donald J. Winzor ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Binding Sites ◽  
Spectrophotometric Titration ◽  
Hydroxamic Acids ◽  
Jack Bean Urease ◽  
Equivalent Weight ◽  
Guanidinium Chloride ◽  
Jack Bean ◽  
Equilibrium Ultracentrifugation

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 ± 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of β-mercaptoethanol is ~95 000. Essentially the same subunit molecular weight (~93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride – β-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein–inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function

1992 ◽  
Vol 72 (4) ◽  
pp. 1611-1615 ◽  
Author(s):  
R. E. Weber
Keyword(s):  
Binding Sites ◽  
Ionic Composition ◽  
Oxygen Affinity ◽  
Chloride Concentration ◽  
Anion Binding ◽  
Chloride Binding ◽  
Ph Values ◽  
Two Temperatures ◽  
Ethanesulfonic Acid ◽  
Chloride Concentrations

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.

scholarly journals Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site

1988 ◽  
Vol 256 (1) ◽  
pp. 279-282 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Reversed Phase ◽  
Binding Activity ◽  
Complete Loss ◽  
Tryptophan Residue ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Biotin Binding ◽  
Peptide Fractions ◽  
Specific Reagent

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.

Biotinylated Polythiophene Copolymer – a Novel Electroactive Biomaterial Utilizing the Biotin-Streptavidin Interaction

1992 ◽  
Vol 292 ◽  
Author(s):  
Jeong-Ok Lim ◽  
Manjunath Kamath ◽  
Kenneth A. Marx ◽  
Sukant K. Tripathy ◽  
David L. Kaplan ◽  
...  
Keyword(s):  
Binding Sites ◽  
Solid Substrate ◽  
Emission Peak ◽  
One Dimensional ◽  
Langmuir Blodgett ◽  
Significant Area ◽  
Pressure Area ◽  
Biotin Binding ◽  
Monolayer Assembly ◽  
Area Expansion

AbstractA novel hierarchical biomaterial capable of incorporating any biotinylated biomolecule has been created. Our strategy is to biotinylate one-dimensional electroactive polymers and use a bridging streptavidin protein on Langmuir-Blodgett (LB) organized films. The following copolymeric system which enables functionalization of other molecules and formation of good monolayers was employed. Biotinylated poly(3-methanolthiophene-co-3-undecylthiophene) (B-PMUT) demonstrated a significantly better isotherm implying superior molecular packing compared to poly(3-methanolthiophene-co-3-undecylthiophene) (PMUT) on the LB airwater surface. The isotherm showed significant area expansion when streptavidin was injected below the B-PMUT monolayer in 0.1mM NaH2PO4/0.1 M NaCl buffer (pH 6.8) subphase. We then incorporated biotinylated phycoerythrin (B-PE) into this novel biomaterial by binding the unoccupied biotin binding sites on the bound streptavidin (4 sites total). The pressure-area isotherm of the protein injected monolayer showed area expansion. A characteristic fluorescent emission peak at 576nm was detected from the monolayer transferred onto a solid substrate. These observations demonstrated the function of B-PMUT in hierarchical monolayer assembly of molecules incorporating the biotin / streptavidin interaction.

scholarly journals Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations

2021 ◽  
Author(s):  
Luning Yu ◽  
Xinqi Kang ◽  
Mohammad Amin Alibakhshi ◽  
Mikhail Pavlenok ◽  
Michael Niederweis ◽  
...  
Keyword(s):  
Guanidinium Chloride ◽  
Polymer Bilayers ◽  
Protein Channel ◽  
Chloride Concentrations

scholarly journals Serological and fluorescence studies of the heat stability of bovine lactoferrin

1979 ◽  
Vol 46 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Augustin Baer ◽  
Marko Oroz ◽  
Bernard Blanc
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Complement Fixation ◽  
Heat Stability ◽  
Heat Denaturation ◽  
Bovine Lactoferrin ◽  
Iron Binding ◽  
Binding Ability ◽  
Fluorescence Studies ◽  
Tryptophan Residues

SUMMARYThe heat denaturation of Fe-saturated lactoferrin (If) and Fe-free lactoferrin (apo-lf) was studied using the methods of micro-complement fixation and fluorescence. It was established that the change in conformation of apo-lf, induced by iron binding, conferred a higher heat stability to the molecule: the changes were observed at temperatures above 40 °C for apo-lf and above 60 °C for If. The Fe-binding ability of the protein was partially independent of the degree of denaturation. Fluorescence analyses indicated that tryptophan residues were probably not directly involved in the metal binding. There was no evidence of antibodies interfering with the binding sites.

Sign in / Sign up

Export Citation Format

Share Document