scholarly journals The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments

1975 ◽  
Vol 149 (1) ◽  
pp. 73-82 ◽  
Author(s):  
S K Dower ◽  
R A Dwek ◽  
A C McLaughlin ◽  
L E Mole ◽  
E M Press ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin G ◽  
Metal Binding ◽  
Binding Sites ◽  
Water Proton ◽  
Proton Relaxation ◽  
Relaxation Rates ◽  
Protein Fluorescence ◽  
Chain Dimer ◽  
Rabbit Igg

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Fab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very ‘tight’ sites on the Fc fragment (Kd approx. 5 μM) and two weaker sites on each Fab region (Kd approx. 140 μM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.

scholarly journals Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli

1994 ◽  
Vol 300 (2) ◽  
pp. 373-381 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Protein Fluorescence ◽  
Metal Binding Sites ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Aminolaevulinic Acid Dehydratase

Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

scholarly journals Interchain disulphide-bond formation in the assembly of immunoglobulin G. Heavy-chain dimer as an intermediate

1968 ◽  
Vol 109 (4) ◽  
pp. 637-643 ◽  
Author(s):  
Brigitte A. Askonas ◽  
Alan R. Williamson
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Immunoglobulin G ◽  
Bond Formation ◽  
Disulphide Bond ◽  
Pulse Labelling ◽  
Myeloma Protein ◽  
Chain Dimer ◽  
Disulphide Bond Formation ◽  
Covalently Linked

1. The role of disulphide-bond formation in the assembly of G2a myeloma protein 5563 was studied by pulse-labelling ascitic plasma cells of tumour-line 5563 for 2–8min. with radioactive amino acids, and analysing the intracellular proteins. Myeloma-protein determinants were first purified by ion-exchange chromatography under conditions that do not dissociate non-covalently linked sub-units of immunoglobulin G. The pulse-labelled material was then analysed by electrophoresis on polyacrylamide gels in sodium dodecyl sulphate–phosphate–urea buffer, which dissociates non-covalently linked sub-units; after gel electrophoresis, radioactive protein bands were located by radioautography, and characterized immunologically after elution. 2. Two heavy-chain intermediates were detected: (i) heavy-chain dimer; (ii) the dimer with one light chain attached. Free light chains had previously been shown to be intermediates in assembly. No evidence for the presence of half-molecules (one light chain attached to one heavy chain) was obtained. The formation of the disulphide bond between the heavy chains thus appears to precede the light-chain–heavy-chain linkage in immunoglobulin G assembly.

scholarly journals Revisiting paramagnetic relaxation enhancements in slowly rotating systems: how long is the long range?

2020 ◽  
Author(s):  
Giovanni Bellomo ◽  
Enrico Ravera ◽  
Vito Calderone ◽  
Mauro Botta ◽  
Marco Fragai ◽  
...  
Keyword(s):  
Chemical Exchange ◽  
Protein Structure Determination ◽  
Bulk Water ◽  
Water Proton ◽  
Proton Relaxation ◽  
Relaxation Rates ◽  
Rotating Systems ◽  
Complete Relaxation ◽  
Nuclear Magnetization ◽  
Order Of Magnitude

Abstract. Cross relaxation terms in paramagnetic systems that reorient rigidly with slow tumbling times can increase the effective longitudinal relaxation rates of protons of more than one order of magnitude. This is evaluated by simulating the time evolution of the nuclear magnetization using a complete relaxation matrix approach. The calculations show that the Solomon dependence of the relaxation rates on the metal-proton distance (as r−6) can be incorrect for protons farther than 15 Å from the metal, and thus can originate sizable errors in R1-derived distance restraints used, for instance, for protein structure determination. Furthermore, the chemical exchange of these protons with bulk water protons can enhance the relaxation rate of the solvent protons by far more than expected from the Solomon equation. Therefore, it may contribute significantly to the water proton relaxation rates measured at MRI magnetic fields in the presence of slow-rotating nanoparticles containing paramagnetic ions and a large number of exchangeable surface protons.

scholarly journals Revisiting paramagnetic relaxation enhancements in slowly rotating systems: how long is the long range?

2021 ◽  
Vol 2 (1) ◽  
pp. 25-31
Author(s):  
Giovanni Bellomo ◽  
Enrico Ravera ◽  
Vito Calderone ◽  
Mauro Botta ◽  
Marco Fragai ◽  
...  
Keyword(s):  
Relaxation Rate ◽  
Chemical Exchange ◽  
Protein Structure Determination ◽  
Bulk Water ◽  
Paramagnetic Relaxation ◽  
Water Proton ◽  
Proton Relaxation ◽  
Relaxation Rates ◽  
Rotating Systems ◽  
Order Of Magnitude

Abstract. Cross-relaxation terms in paramagnetic systems that reorient rigidly with slow tumbling times can increase the effective longitudinal relaxation rates of protons of more than 1 order of magnitude. This is evaluated by simulating the time evolution of the nuclear magnetization using a complete relaxation rate-matrix approach. The calculations show that the Solomon dependence of the paramagnetic relaxation rates on the metal–proton distance (as r−6) can be incorrect for protons farther than 15 Å from the metal and thus can cause sizable errors in R1-derived distance restraints used, for instance, for protein structure determination. Furthermore, the chemical exchange of these protons with bulk water protons can enhance the relaxation rate of the solvent protons by far more than expected from the paramagnetic Solomon equation. Therefore, it may contribute significantly to the water proton relaxation rates measured at magnetic resonance imaging (MRI) magnetic fields in the presence of slow-rotating nanoparticles containing paramagnetic ions and a large number of exchangeable surface protons.

scholarly journals The interaction of protein A and Fc fragment of rabbit immunoglobulin G as probed by complement-fixation and nuclear-magnetic-resonance studies

1977 ◽  
Vol 167 (3) ◽  
pp. 661-668 ◽  
Author(s):  
C Wright ◽  
K J Willan ◽  
J Sjödahl ◽  
D R Burton ◽  
R A Dwek
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Immunoglobulin G ◽  
Complement Fixation ◽  
Protein A ◽  
Structural Basis ◽  
Proton Relaxation ◽  
Fc Fragment ◽  
Rabbit Igg ◽  
Nuclear Magnetic

Protein-A-Fc-fragment complexes were observed in sedimentation-velocity experiments by ultracentrifugation. The interaction was studied by protein-fluorescence-quenching titrations of the Fc fragment with protein A, allowing the dissociation constant to be determined under a variety of conditions. The first component of the complement pathway, C1, is activated by complexes of protein A with rabbit IgG (immunoglobulin G), and the structural basis for this interaction was studied by using n.m.r. (nuclear magnetic resonance). The four Fc-fragment binding sites on protein A were shown to contain aromatic amino acids, and to be connected by mobile hydrophilic regions. Neither n.m.r. nor proton-relaxation-enhancement studies show evidence of a large conformational change of the Fc fragment on binding protein A, and this suggests that the cross-linking of the Fc fragments may be primarily responsible for the activation of component C1. This is supported by the inability of a univalent tryptic fragment of protein A to activate complement fixation by rabbit IgG.

Modulation of water proton relaxation rates by liposomes containing paramagnetic materials

1988 ◽  
Vol 6 (4) ◽  
pp. 445-458 ◽  
Author(s):  
G. Bačić ◽  
M. R. Niesman ◽  
H. F. Bennett ◽  
R. L. Magin ◽  
H. M. Swartz
Keyword(s):  
Water Proton ◽  
Proton Relaxation ◽  
Relaxation Rates ◽  
Paramagnetic Materials

scholarly journals Differences in the protein fluorecence of the two iron(III)-binding sites of ovotransferrin

1975 ◽  
Vol 145 (2) ◽  
pp. 201-207 ◽  
Author(s):  
R W Evans ◽  
J J Holbrook
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Electronic Excitation ◽  
Tryptophan Fluorescence ◽  
Electronic Excitation Energy ◽  
Visible Absorption Spectrum ◽  
Protein Fluorescence ◽  
Visible Absorption ◽  
Metal Binding Sites ◽  
Stable Hybrid

1. Changes in the tryptophan fluorescence and the visible absorption spectrum resulting from the combination of apo-ovotransferrin with Fe3+, F,E2+, Cu2+, Zn2+, Mn2+, and Cd2+were measured. 2. As expected for a radiationless transfer of electronic excitation energy, only the ions Fe3+, Fe2+and Cu2+, which gave complexes with large extinctions between 300 and 370nm, resulted in large decreases in trytophan fluorescence. 3. The decrease in protein fluorescence was non-linear with increasing occupancy of the Fe3+ -and Cu2+ - binding sites. The decrease in fluorescence on binding of Fe3+ was biphasic and showed that the two metal-binding sites were being occupied sequentially at pH7.4-8.4. The first site reacted with Fe3+ instantaneously, the second was occupied over a minute. 5. The nonidentity of the two sites was also demonstrated by the preparation of a stable hybrid containing both Cu2+ and Zn2+.h Cu2+ and Zn2+

Sign in / Sign up

Export Citation Format

Share Document