scholarly journals The Effect of Reduction of The Disulphide Bond on the Fluorescence of High-Sulphur Wool Proteins

1968 ◽  
Vol 21 (6) ◽  
pp. 1325 ◽  
Author(s):  
GM Bhatnagar ◽  
LC Gruen
Keyword(s):  
Protein Conformation ◽  
Fluorescence Emission ◽  
Disulphide Bond ◽  
Thiol Groups ◽  
Quenching Effect ◽  
Tyrosine Fluorescence ◽  
Disulphide Bonds ◽  
Wool Proteins ◽  
Made In ◽  
Model Peptides

Reduction of the disulphide bonds affects the fluorescence emission from many proteins; this change is generally correlated with a change in the protein conformation (Cowgill 1964, 1966; Stryer, Holmgren, and Reichard 1967). Cowgill (1967) has reported the quenching effect of disulphide and thiol groups on the tryptophan and tyrosine fluorescence from model peptides when the quenching moiety is present in the same molecule as the chromophore. Similar observations have been made in this laboratory using a high-sulphur fraction extracted from wool protein.

scholarly journals Extraction of Reduced Wool Proteins by Solutions of Salts

1969 ◽  
Vol 22 (4) ◽  
pp. 1081 ◽  
Author(s):  
JA Maclaren ◽  
DJ Kilpatrick
Keyword(s):  
Aqueous Solution ◽  
Solvent System ◽  
Disulphide Bonds ◽  
Wool Proteins

It has been shown earlier that the disulphide bonds in wool fibres are reduced specifically and almost quantitatively in aqueous solution using toluene-w-thiol (Maclaren 1962) or tributyl phosphine (Sweetman and Maclaren 1966; Maclaren, Kilpatrick, and Kirkpatrick 1968). The next step was to find a suitable solvent system to extract the proteins from the reduced fibre.

scholarly journals Cysteine immobilisation on the polyethylene terephthalate surfaces and its effect on the haemocompatibility

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Balaji Ramachandran ◽  
Vignesh Muthuvijayan
Keyword(s):  
Polyethylene Terephthalate ◽  
Disulphide Bond ◽  
Stability Studies ◽  
Short Term ◽  
Amide Bonds ◽  
Disulphide Bonds ◽  
Disulphide Bond Formation ◽  
Signalling Molecule ◽  
No Release ◽  
Important Signalling Molecule

Abstract Nitric oxide (NO) is an important signalling molecule involved in haemostasis. NO, present as endogenous S-nitrosothiols, is released by cysteine through a transnitrosation reaction. To exploit this mechanism, cysteine was immobilised onto the different carboxylated polyethylene terephthalate (PET) surfaces using 1-step EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) crosslinking mechanism. Immobilised cysteine concentration and NO release were dependent on the surface carboxyl density. Stability studies showed that the immobilised cysteine concentration and NO release reduced within 6 h. Immobilisation of cysteine derivatives eliminated the possibility of formation of polycysteine and its electrostatic interaction with the carboxylated PET. The immobilised cysteine concentration did not recover after DTT treatment, eliminating the possibility of disulphide bond formation. Further, cysteine was immobilised using a 2-step EDC crosslinking mechanism. Although the cysteine concentration reduced during stability studies, it recovered upon DTT treatment, indicating that cysteine forms amide bonds with the carboxylated PET and the observed decrease in cysteine concentration is probably due to the formation of disulphide bonds. The haemocompatibility of the cysteine immobilised PET surfaces showed similar results compared to the carboxylated PET. The loss of thiol groups due to the disulphide bond restricts the transnitrosation reaction. Hence, these materials can be used primarily in short-term applications.

Fluorescence Emission Behavior of Eu(III) Sorbed on Calcium Silicate Hydrates Formed With No Dried Process

2013 ◽  
Author(s):  
Yuichi Niibori ◽  
Masayuki Narita ◽  
Akira Kirishima ◽  
Taiji Chida ◽  
Hitoshi Mimura
Keyword(s):  
Energy Transfer ◽  
Calcium Silicate ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Fluorescence Emission Spectra ◽  
Light Water ◽  
Quenching Effect ◽  
Emission Behavior ◽  
Main Component ◽  
The Eu

Calcium silicate hydrate (CSH) is a main component of cement-based material required for constructing the geological repository. As in many countries, since the repository in Japan is constructed below water table, we must consider the interaction of radionuclide with cement materials altered around the repository after the backfill. Using fluorescence emission spectra, so far, the authors have investigated the interaction of Eu(III) (as a chemical analog of Am(III)) with CSH gels formed with no dried process, considering a condition saturated with groundwater. However, in such fluorescence emission behaviors, a deexcitation process of OH vibrators of light water and a quenching effect caused by Eu-Eu energy transfer between Eu atoms incorporated in the CSH gel must be considered. This study examined the fluorescence emission behavior of Eu(III) sorbed on CSH gels formed with no dried process, by using La(III) (non-fluorescent ions) as a diluent of Eu(III). Furthermore, the CSH samples were synthesized with CaO, SiO2, and heavy water (D2O) as a solvent in order to avoid the obvious deexcitation process of OH vibrators of light water. This study prepared CSH samples with the Ca/Si ratio set to 1.6, 1.0, and 0.5. A 1 mM solution of a given combination of Eu(III) and La(III) (Eu(III) content: 100%, 67%, 50% or 33%) was added into CSH gel sample. The contact time-period of the CSH gel with the Eu(III)/La(III) solution was set to 60 days. In the results, the peak around 618 nm was split into two peaks of 613 nm and 622 nm in the cases of Ca/Si = 1.0 and 1.6. Then, the peak of 613 nm decreased with increment of Eu(III)/La(III) ratio. This means that the relative intensity of 613 nm is useful to quantify the amount of Eu(III) incorporated in CSH gel. Besides, the intensity peak of 584 nm decayed with increment of Eu/La ratio, suggesting a quenching effect due to Eu-Eu energy transfer. However, the decay behavior of the fluorescence emission did not depend on the Eu/La concentration ratio. That is, such a quenching effect is neglectable. Additionally, the low Ca/Si ratio samples underwent slow attenuation of fluorescence and showed profiles similar to those of high Ca/Si ratio samples. Therefore, low Ca/Si ratio samples also include the reaction forming a complex on the surface of CSH gel with Eu(III). In other words, even if Ca/Si ratio is lower than 1.0, CSH gels would retard the migration of radionuclides released from the repository.

scholarly journals The disulphide bond structure of thyroid-stimulating hormone β-subunit

1996 ◽  
Vol 314 (2) ◽  
pp. 449-455 ◽  
Author(s):  
W. Douglas FAIRLIE ◽  
Peter G. STANTON ◽  
Milton T. W. HEARN
Keyword(s):  
Peptide Mapping ◽  
Reversed Phase ◽  
Thyroid Stimulating Hormone ◽  
Exchange Chromatography ◽  
Disulphide Bond ◽  
Double Labelling ◽  
Β Subunit ◽  
Reversed Phase Hplc ◽  
Reactive Group ◽  
Disulphide Bonds

Previously only one of the six disulphide bonds within the β-subunit of bovine thyrotropin (bTSHβ) has been unequivocally assigned. In the present investigation, the fluorescent alkylating reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid has been employed as part of a double-alkylation strategy to allow the relative reactivities and the location of the six disulphide bonds of bTSHβ, after selective reduction, to be assigned by using reversed-phase HPLC peptide mapping techniques and associated methods of structural analysis. The most reactive disulphide bond was Cys88–Cys95; the second most reactive group of disulphide bonds involved the half-cystine residues Cys16, Cys19, Cys67 and Cys105 with the experimental results consistent with the assignment of disulphide bonds to Cys16–Cys67 and Cys19–Cys105. The least reactive group of half-cystine residues consisted of Cys2, Cys27, Cys31, Cys52, Cys83 and Cys85. The isolation, by high-performance ion-exchange chromatography, of a partly reduced bTSHβ derivative in which only the half-cystine residues Cys31, Cys85, Cys88 and Cys95 were labelled enabled the assignment of a previously uncharacterized disulphide bond to Cys31–Cys85. The remaining two assignments, Cys2–Cys52 and Cys27–Cys83, were made by comparison with the recently published human chorionic gonadotropin crystal structure. The flexibility of the double-labelling approach used in these studies demonstrates that only very small quantities are required for proteins containing an extensive number of half-cystine residues such as TSHβ, owing to the combination of the high resolution of the reversed-phase HPLC peptide mapping procedures and the sensitivity of the fluorimetric detection method.

The Escherichia coli CcmG protein fulfils a specific role in cytochrome c assembly

2001 ◽  
Vol 355 (1) ◽  
pp. 51-58 ◽  
Author(s):  
Eleanor REID ◽  
Jeff COLE ◽  
Deborah J. EAVES
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Redox State ◽  
Disulphide Bond ◽  
Anaerobic Growth ◽  
Terminal Electron Acceptor ◽  
Bond Forming ◽  
Disulphide Bonds ◽  
Low Concentrations ◽  
K 12

In Escherichia coli K-12, c-type cytochromes are synthesized only during anaerobic growth with trimethylamine-N-oxide, nitrite or low concentrations of nitrate as the terminal electron acceptor. A thioredoxin-like protein, CcmG, is one of 12 proteins required for their assembly in the periplasm. Its postulated function is to reduce disulphide bonds formed between correctly paired cysteine residues in the cytochrome c apoproteins prior to haem attachment by CcmF and CcmH. We report that loss of CcmG synthesis by mutation was not compensated by a second mutation in disulphide-bond-forming proteins, DsbA or DsbB, or by the chemical reductant, 2-mercaptoethanesulphonic acid. An anti-CcmG polyclonal antibody was used in Western-blot analysis to probe the redox state of CcmG in mutants defective in the synthesis of other proteins essential for cytochrome c assembly. The oxidized form of CcmG accumulated not only in trxA or dipZ mutants defective in the transfer of electrons from the cytoplasm for disulphide isomerization and reduction reactions in the periplasm, but also in ccmF and ccmH mutants. The requirement of both CcmF and CcmH for the reduction of the disulphide bond in CcmG indicates that CcmG functions later than CcmF and CcmH in cytochrome c assembly, rather than in electron transfer from the membrane-associated DipZ (also known as DsbD) to CcmH. The data support a model proposed by others in which CcmG catalyses one of the last reactions specific to cytochrome c assembly.

scholarly journals Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase

1991 ◽  
Vol 274 (3) ◽  
pp. 781-786 ◽  
Author(s):  
M F Fillat ◽  
D E Edmondson ◽  
C Gomez-Moreno
Keyword(s):  
Anabaena Variabilis ◽  
Disulphide Bond ◽  
Activation Process ◽  
Thiol Groups ◽  
Native Form ◽  
Steady State Kinetic ◽  
Significant Difference ◽  
Cyanobacterium Anabaena ◽  
State Kinetic

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.

scholarly journals The effect of chemical modifications induced in insulin on the reactivity of the interchain disulphide bonds towards sodium sulphite

1968 ◽  
Vol 108 (2) ◽  
pp. 247-255 ◽  
Author(s):  
A. Massaglia ◽  
F. Pennisi ◽  
U. Rosa ◽  
S. Ronca-Testoni ◽  
C. A. Rossi
Keyword(s):  
Biological Activity ◽  
Mode Of Action ◽  
High Reactivity ◽  
Sodium Sulphite ◽  
Thiol Groups ◽  
Chemical Modifications ◽  
Amperometric Titration ◽  
Disulphide Bonds ◽  
Native Insulin

The reactivity of the three disulphide bridges of insulin towards sodium sulphite was studied by amperometric titration of the liberated thiol groups. In the native, acetylated or succinylated molecule two bridges react at pH7, but in the methylated or phenylcarbamoylated molecule only one bridge reacts. All three bridges react in all derivatives in 8m-urea or at pH9. Loss in biological activity parallels the loss in reactivity of one of the bridges during methylation. It is suggested that change in reactivity of the S·S bonds reflects the occurrence of a conformational modification of the protein. The possibility is discussed that the unusually high reactivity of the S·S bonds in native insulin depends strictly on the integrity of the native molecule, suggesting that S·S bonds are in some way involved in the hormone's mode of action.

scholarly journals Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine

1983 ◽  
Vol 215 (3) ◽  
pp. 513-518 ◽  
Author(s):  
A R S Prasad ◽  
J Ybarra ◽  
J S Nishimura
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Adenine Nucleotide ◽  
Bond Formation ◽  
Disulphide Bond ◽  
Thiol Groups ◽  
Binding Region ◽  
Nucleotide Analogue ◽  
Disulphide Bond Formation ◽  
Succinyl Coa Synthetase

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine (5′-FSBA). Optimal inactivation by 5′-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5′-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5′-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5′-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5′-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5′-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5′-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.

scholarly journals Polycyclic Aromatic Hydrocarbon Solute Probes: Effect of Solvent Polarity on the Ovalene and Benzo[ghi]perylene Fluorescence Emission Fine Structures

1988 ◽  
Vol 42 (8) ◽  
pp. 1525-1531 ◽  
Author(s):  
Riaz Waris ◽  
Michael A. Rembert ◽  
David M. Sellers ◽  
William E. Acree ◽  
Kenneth W. Street ◽  
...  
Keyword(s):  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Fluorescence Emission ◽  
Solvent Polarity ◽  
Fluorescence Properties ◽  
Effect Of Solvent ◽  
Fluorescence Measurements ◽  
Polycyclic Aromatic ◽  
Fine Structures ◽  
Made In

Fluorescence properties of benzo[ghi]perylene (BPe) and ovalene (Ov) dissolved in 25 solvents of varying polarity are reported. Measurements indicate that emission intensities depend on solvent polarity. The BPe and Ov solvent polarity scales are defined as the ratio of the fluorescence emission intensities of bands I and III of the vibronic spectra. Benzo[ghi]perylene and ovalene solute probes enable fluorescence measurements to be made in spectral regions less prone to solvent inner filtering and other artifacts which have hampered the use of pyrene (Py) as a polarity probe molecule.

scholarly journals Rapid and selective modification of phosphoserine residues catalysed by Ba2+ ions for their detection during peptide microsequencing

1991 ◽  
Vol 280 (1) ◽  
pp. 261-265 ◽  
Author(s):  
M F Byford
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Metal Ions ◽  
Ion Concentration ◽  
Thiol Groups ◽  
Disulphide Bonds ◽  
Rate Of Reaction ◽  
Alkaline Conditions ◽  
Beta Elimination ◽  
Alpha Beta

The beta-elimination of phosphoserine residues by dilute alkali is catalysed by the presence of group II metal ions. The use of 0.1 M-Ba (OH)2 catalysed the rate of beta-elimination of phosphoserine by more than two orders of magnitude compared with the use of NaOH at the same OH-ion concentration. Serine and threonine residues are unaffected by this treatment. Free thiol groups and disulphide bonds are labile to these conditions, but carboxymethylcysteine is stable. The rate of beta-elimination of O-glycosidically linked moieties is not catalysed under these conditions, and the rate of reaction is thus two orders of magnitude slower than for phosphoserine. This specific catalysis was readily exploited in the rapid and selective modification of phosphoserine residues under mildly alkaline conditions with the nucleophile methylamine via the alpha beta-desaturated dehydroalanine intermediate to yield the beta-methylaminoalanine residue. This modified residue could be easily detected on sequence analysis and in amino acid compositions.

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