Reaction of Fluorescein Isothiocyanate with Thiol and Amino Groups of Sarcoplasmic ATPase

1985 ◽  
Vol 40 (11-12) ◽  
pp. 863-875 ◽  
Author(s):  
Gertrude Swoboda ◽  
Wilhelm Hasselbach
Keyword(s):  
Absorption Maximum ◽  
Sodium Dodecylsulfate ◽  
Specific Effect ◽  
Fluorescein Isothiocyanate ◽  
Thiol Groups ◽  
Amino Groups ◽  
Model Compounds ◽  
Intramolecular Transfer ◽  
Dodecyl Ether ◽  
Lysyl Residue

Abstract Several model compounds containing thiol and/or amino groups (mercaptoethanol, glutathione, cysteine, ethanolamine, glycine) were studied with respect to their reactivity towards fluorescein isothiocyanate (followed spectrophotometrically at 504 and 412 nm), stability of product and long­ wave absorption maximum of the fluorescein residue attached. Thiol groups reacted by far more readily than amino groups. A specific effect was observed with cysteine, indicating an intramolecular transfer of the fluorescein residue from SH to NH2.With sarcoplasmic vesicles both types of reactions were observed. The ratio of products, which can be distinguished by their different stabilities and absorption spectra, depended on the absence or presence of detergents. While with native vesicles the NH2 reaction predominated, with vesicles solubilized with sodium dodecylsulfate, octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine the SH reaction became prevailing. Already 0.35 mg sodium dodecylsulfate per mg protein were sufficient to give rise to dithiourethane formation exclusively. Excess fluorescein isothiocyanate reacted with several thiol groups of dodecylsulfate-solubilized vesicles. In the presence of ATP binding of fluorescein isothiocyanate to native vesicles was significantly reduced.Total blockage of the vesicular SH groups with N-ethyl-maleimide led to preparations that reacted with fluorescein isothiocyanate much more slowly, compared to native vesicles. Octaethy­ leneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine in the assay accelerated the thioureide formation from N-ethylmaleimide modified vesicles, whereas sodium dodecylsulfate prevented it almost completely.Our results support the suggestion that one or several thiol groups in vicinity of the highly reactive lysyl residue might play a role in the fast specific reaction, which is only observed with intact native vesicles.

scholarly journals The measurement of amino groups in proteins and peptides

1971 ◽  
Vol 124 (3) ◽  
pp. 581-590 ◽  
Author(s):  
R Fields
Keyword(s):  
Amino Acids ◽  
Room Temperature ◽  
Thiol Group ◽  
Amino Group ◽  
Thiol Groups ◽  
Amino Groups ◽  
Model Compounds ◽  
Time Required ◽  
Short Time ◽  
Proteins And Peptides

A technique is examined for determining amino groups with 2,4,6-trinitrobenzenesulphonic acid, in which the extinction at 420nm of sulphite complexes of the trinitrophenylated amino groups is measured. The sensitivity of the method is 5–200nmol of amino group. The method is especially suitable for checking the extent of blocking or unblocking of amino groups in proteins and peptides, owing to the short time required for reaction (5min at room temperature). The reaction of the reagent with thiol groups has been studied and was found to proceed 30–50 times faster than with ∈-amino groups of model compounds. The ∈420 of a trinitrophenylated thiol group was found to be 2250m-1·cm-1. The reaction with several amino acids, peptides and proteins is presented. The ∈420 of a typical α-amino group was found to be 22000m-1·cm-1 and that of an ∈-amino group, 19200m-1·cm-1. Difficulties inherent in the analysis of constituent amino group reactions in proteins are discussed.

Self-hydrogen transfer hydrogenolysis of β-O-4 linkages in lignin catalyzed by MIL-100(Fe) supported Pd–Ni BMNPs

2017 ◽  
Vol 19 (19) ◽  
pp. 4538-4543 ◽  
Author(s):  
Jia-wei Zhang ◽  
Guo-ping Lu ◽  
Chun Cai
Keyword(s):  
Hydrogen Transfer ◽  
Catalytic Performance ◽  
Model Compounds ◽  
Lignin Model Compounds ◽  
Organosolv Lignin ◽  
Lignin Model ◽  
Intramolecular Transfer ◽  
Supported Pd

A MIL-100(Fe) supported Pd–Ni BMNP catalyst has been fabricated, and the catalyst exhibits superior catalytic performance toward intramolecular transfer hydrogenolysis of lignin model compounds and organosolv lignin.

Kinetics of the Reaction of Nitrous Acid with Model Compounds and Proteins, and the Conformational State of N-terminal Groups in the Chymotrypsin Family

1972 ◽  
Vol 50 (12) ◽  
pp. 1282-1296 ◽  
Author(s):  
A. Kurosky ◽  
T. Hofmann
Keyword(s):  
Nitrous Acid ◽  
Conformational State ◽  
Serine Proteinases ◽  
Amino Groups ◽  
Model Compounds ◽  
Third Order ◽  
Tyrosine Residues ◽  
Reactive Groups ◽  
Primary Amino ◽  
Kinetics Of

The kinetics of the reaction of nitrous acid at 4° and pH 4.0 with various amino acids, peptides, and proteins were studied. The reaction with isoleucine methyl ester was found to have a linear dependence on the square of the HONO concentration showing that N2O3 was the reactive species. Third order nitrosation rate constants of primary amino groups showed a correlation with their pK values. They were calculated for the concentration of the unprotonated species to give intrinsic reactivities. The rate of nitrosation of acetyltryptophan to give N-nitrosoacetyltryptophan was found to be a linear function of the nitrous acid concentration. This nitrosation therefore follows a different mechanism. The reaction of nitrous acid with tyrosine residues was examined by spectrophotometry. The reaction was negligible compared to that of other groups. Acetylhistidine and imidazole did not react. Reactivities for α-amino groups, ε-amino groups, and other residues in proteins were compared. The conformational state of the N-terminal residues in serine proteinases, as revealed from their reactivities, is discussed in detail. It is concluded that nitrous acid reacts preferentially with "surface" residues and is a useful tool for exploring conformational states of reactive groups in proteins, especially α-amino groups and indole rings.

scholarly journals The reaction of aldolase with 2-methylmaleic anhydride

1970 ◽  
Vol 116 (5) ◽  
pp. 843-849 ◽  
Author(s):  
I. Gibbons ◽  
R. N. Perham
Keyword(s):  
Enzyme Activity ◽  
Maleic Anhydride ◽  
Enzymic Activity ◽  
Side Reactions ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Amino Groups ◽  
Partial Loss ◽  
Reaction Proceeds ◽  
Citraconic Anhydride

1. The reaction of rabbit muscle aldolase with 2-methylmaleic anhydride is described. All the protein amino groups can be reversibly blocked. 2. As the reaction proceeds, the enzyme activity decreases until, at about 50% citraconylation of amino groups, the enzyme is completely inhibited. At this stage, little or no dissociation of the enzyme tetramer is observed and 75% of the activity is recoverable on unblocking the amino groups. 3. At 80% blocking, the enzyme is completely dissociated but little enzymic activity is recoverable after unblocking. Inability to recover activity after citraconylation and unblocking correlates with the onset of dissociation of the citraconyl-aldolase seen on ultracentrifugation. 4. The only irreversible modification of the enzyme primary structure detectable after the citraconylation and unblocking reactions is the partial loss of thiol groups. It is probable that this is responsible for the inability to reform active enzyme from the citraconylated subunit. 5. Other reversible side reactions of maleic anhydride and citraconic anhydride that may occur with proteins are discussed.

BINDING OF ZINC TO INSULIN AND SOME INSULIN DERIVATIVES STUDIED BY PAPER ELECTROPHORESIS

1969 ◽  
Vol 61 (3) ◽  
pp. 561-576 ◽  
Author(s):  
K. Brunfeldt ◽  
B. A. Hansen ◽  
J. Hoiriis Nielsen
Keyword(s):  
Binding Capacity ◽  
Fluorescein Isothiocyanate ◽  
Paper Electrophoresis ◽  
Zinc Binding ◽  
Amino Groups ◽  
Histidine Residues ◽  
Tyrosine Residues ◽  
Barbital Buffer ◽  
Pronounced Reduction ◽  
Electrophoretic Fractionation

ABSTRACT Paper electrophoretic fractionation in barbiturate (barbital) buffer, pH 9, of iodine-substituted insulin 0–10.8 I/mole showed that substitution with 4–6 I/mole influences the binding of zinc to a demonstrable extent. The effect appears to be due to substitution in the imidazole groups of the histidine residues. Substitution with iodine in the tyrosine residues seems to be without significance, at least at the lower degrees of iodination. The importance of the histidine residues for the binding of zinc is shown by selective destruction of the imidazole groups by photo-oxidation, sensitized by methylene blue. Carbamylation of the N-terminal α-amino groups in the A- and B-chains with KOCN only slightly influences the ability to bind zinc while carbamylation with fluorescein isothiocyanate in the N-terminal of the B-chain brings about a more pronounced reduction in the zinc binding capacity.

scholarly journals Untersuchungen über die Bindung pflanzlicher Wuchsstoffe an verschiedene Komponenten des Chromatins in vitro. I. Bindung an DNS / Investigations Upon the Binding of Plant Growth Substances to Several Components of the Chromatin in vitro. I. Binding to DNA

1971 ◽  
Vol 26 (6) ◽  
pp. 607-612 ◽  
Author(s):  
Günter Fellenberg
Keyword(s):  
Hydrogen Bonds ◽  
Absorption Maximum ◽  
Double Helix ◽  
Specific Effect ◽  
Growth Substance ◽  
Growth Substances ◽  
Plant Growth Substances ◽  
The Stability ◽  
Dna Double Helix

At pH 9,5 the DNA absorption maximum at 190 nm was shifted to 212 nm. This absorption maximum showed a bathochrome effect in the presence of IAA, GA and KI. The amplitude of this maximum was reduced at the same time. By addition of urea (0.1 —2.0 moles/l) the bathochrome effect, induced by the growth substances, was completely reversed, whereas application of NaCl (0.1 — 2.0 moles/l) did not affect the bathochrome movement of this DNA maximum. At pH 6.0 in the presence of 0.9% NaCl this DNA maximum did not show any visible bathochrome movement in the presence of the growth substances investigated. The DNA maximum at 260 nm did not show any alteration in the presence of growth substances.The thermal denaturation of DNA-complexes with IAA and GA showed, that with increasing concentration of these growth substances, the Tm-value of the DNA was reduced. On the other hand, KI increased the Tm-point of DNA. By biphasic melting at 260 nm and 280 nm no specific effect of the investigated growth substances on the stability of the A — T or G—C pairs was detected.Tryptophan and β-NAA closely related in structure to the auxins IAA and α-NAA did not reduce the Tm-value of DNA.At pH < 7 IAA, GA and KI did not show any detectable influence on the Tm-value of DNA.The data presented show that at a pH > 7 IAA, GA and KI are obviously bound to DNA by hydrogen bonds. There is evidence that these bonds are very unstable. Nevertheless, IAA and GA can loosen part of the hydrogen bonds of DNA double helix while KI cannot. Possible consequences of these growth substance effects on DNA are discussed.

Studies in chrome mordanting. II. The binding of chromium(III) cations to wool

1968 ◽  
Vol 21 (11) ◽  
pp. 2723 ◽  
Author(s):  
FR Hartley
Keyword(s):  
Carboxyl Groups ◽  
Thiol Groups ◽  
Amino Groups

The reactions of a number of chromium(111) salts with wool have been investigated. It was found that chromium(111) was not displaced from wool by washing in water. Chromium(111) was bound to wool by the carboxyl groups. Other groups such as sulphonate and thiol groups also bound chromium(111) when these were present, but no evidence was found for binding by amino groups. A mechanism is suggested for the reaction of chromium(111) with wool.

scholarly journals The reversible reaction of protein amino groups with exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride. The reaction with lysozyme

1970 ◽  
Vol 118 (5) ◽  
pp. 733-739 ◽  
Author(s):  
M. Riley ◽  
R. N. Perham
Keyword(s):  
Free Amino ◽  
Tertiary Structure ◽  
Complete Recovery ◽  
Enzymic Activity ◽  
Carboxyl Group ◽  
Amino Group ◽  
Half Time ◽  
Amino Groups ◽  
Model Compounds ◽  
Anchimeric Assistance

1. The reaction of exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride with amino groups of model compounds and lysozyme is described. 2. Reaction with the ∈-amino group of Nα-acetyl-l-lysine amide gives rise to two diastereoisomeric products; at acid pH the free amino group is liberated with anchimeric assistance by the neighbouring protonated carboxyl group with a half-time of 4–5h at pH3.0 and 25°C. 3. The amino groups of lysozyme can be completely blocked, with total loss of enzymic activity. Dialysis at pH3.0 results in complete recovery of the native primary and tertiary structure of lysozyme and complete return of catalytic activity. 4. The specificity of reaction of this and other anhydrides with amino groups in proteins is discussed.

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