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.

scholarly journals o-Quinones formed in plant extracts. Their reactions with amino acids and peptides

1969 ◽  
Vol 112 (5) ◽  
pp. 609-616 ◽  
Author(s):  
W. S. Pierpoint
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plant Extracts ◽  
Thiol Group ◽  
Amino Group ◽  
Amino Groups ◽  
Terminal Amino Acid ◽  
Secondary Reactions ◽  
Terminal Amino ◽  
Group 5

1. The reactions of amino acids and peptides with the o-quinones produced by the enzymic oxidation of chlorogenic acid and caffeic acid have been studied manometrically and spectrophotometrically. 2. Amino acids, except lysine and cysteine, react primarily through their α-amino groups to give red or brown products. These reactions, which compete with the polymerization of the quinones, are followed by secondary reactions that may absorb oxygen and give products with other colours. 3. The ∈-amino group of lysine reacts with the o-quinones in a similar way. The thiol group of cysteine reacts with the quinones, without absorbing oxygen, giving colourless products. 4. Peptides containing cysteine react with the o-quinones through their thiol group. 5. Other peptides, such as glycyl-leucine and leucylglycine, react primarily through their α-amino group and the overall reaction resembles that of the N-terminal amino acid except that it is quicker. 6. With some peptides, the secondary reactions differ from those that occur between the o-quinones and the N-terminal amino acids. The colours produced from carnosine resemble those produced from histidine rather than those from β-alanine, and the reactions of prolylalanine with o-quinones are more complex than those of proline.

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.

A Raman spectroscopic study of the complexation of the methylmercury(II) cation by amino acids

1987 ◽  
Vol 65 (3) ◽  
pp. 491-496 ◽  
Author(s):  
Serge Alex ◽  
Rodrigue Savoie
Keyword(s):  
Amino Acids ◽  
Spectroscopic Investigation ◽  
Thiol Group ◽  
Amino Groups ◽  
Raman Spectroscopic ◽  
Raman Spectroscopic Study ◽  
Bending Mode ◽  
Stretching Vibration ◽  
Vibrational Bands ◽  
Cation Coordination

A systematic Raman spectroscopic investigation of the complexation of CH3Hg+ by the standard amino acids is reported. It is shown that the vibrational bands due to the ligand—Hg and Hg—CH3 stretching modes and to the symmetric —CH3 bending mode of the —HgCH3 unit are well suited to characterize the extent of complexation and the sites of attachment of the cation. Coordination, which occurs mostly on sulfur and nitrogen atoms by substitution of a proton on the thiol group of cysteine or on amino groups in general, is best identified by the frequency of the ligand—Hg stretching vibration in the 250–550 cm−1 region of the spectrum.

Increased Resistance of Peptides to Serum Proteases by Modification of their Amino Groups

2003 ◽  
Vol 58 (7-8) ◽  
pp. 558-561 ◽  
Author(s):  
Rossella Galati ◽  
Alessandra Verdina ◽  
Giuliana Falasca ◽  
Alberto Chersi
Keyword(s):  
Amino Acids ◽  
Proteolytic Enzymes ◽  
Living Organism ◽  
Amino Group ◽  
Amino Groups ◽  
Protein Fragments ◽  
Beta Alanine ◽  
Synthetic Protein ◽  
In Vivo Tests

Abstract The ability of synthetic protein fragments to survive the degradative action of aminopeptidases and serum proteolytic enzymes can be remarkably enhanced by slight modifications at their N-terminal alpha-amino group. This can be achieved by addition of beta-alanine or amino acids of the d-configuration, amino acids which are seldom found in a living organism. These modifications do scarcely modify the chemical and physical properties of the peptides, and should be preferrred, especially for in vivo tests, to drastic alterations of peptides as produced by dinitrophenylation or dansylation of the amino groups.

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.

scholarly journals Glutamine: precursor or nitrogen donor for citrulline synthesis?

2010 ◽  
Vol 299 (1) ◽  
pp. E69-E79 ◽  
Author(s):  
Juan C. Marini ◽  
Inka Cajo Didelija ◽  
Leticia Castillo ◽  
Brendan Lee
Keyword(s):  
Amino Acids ◽  
Current Literature ◽  
Ammonium Acetate ◽  
Carbon Skeleton ◽  
Amino Group ◽  
Amino Groups ◽  
Amido Group ◽  
Nitrogen Donor ◽  
Citrulline Synthesis

Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton vs. nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, l-[2-15N]- and l-[5-15N]glutamine and 15N-ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled the three nitrogen groups of citrulline almost equally. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the δ-nitrogen. D5-glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was utilized poorly, accounting for only 0.2–0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25-fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-13Cn tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for ∼40% of the circulating citrulline. Proline contribution was minor (3.4%), and glutamine was negligible (0.4%). However, the glutamine tracer resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.

Possible reactions of 1,2-naphthaquinone in the eye

1967 ◽  
Vol 102 (3) ◽  
pp. 853-863 ◽  
Author(s):  
R.R. Jancis ◽  
Pirie Antoinette
Keyword(s):  
Amino Acids ◽  
Ascorbic Acid ◽  
Aqueous Humour ◽  
Thiol Group ◽  
Amino Group ◽  
Reaction Products ◽  
Protein Thiol ◽  
Alpha Crystallin ◽  
Group 4 ◽  
Normal Lens

1. Reactions of 1,2-naphthaquinone with amino acids, glutathione and proteins of the lens have been studied in connexion with investigations of naphthalene-induced cataract. 2. Cysteine reacts probably through its amino group with 1,2-naphthaquinone to form either purple or brown compounds with characteristic absorption spectra. 3. Glutathione reacts with 1,2-naphthaquinone through its thiol group. 4. Spectroscopic evidence suggests that 1,2-naphthaquinone reacts with the amino group of amino acids. This reaction may take place in the aqueous humour. 5. The proteins of lens react with 1,2-naphthaquinone to form brown compounds. 6. There is loss of protein thiol in this reaction and the products are less easily digestible by pancreatin than normal lens proteins. 7. The compound of alpha-crystallin and 1,2-naphthaquinone is soluble at neutrality, but the compounds of beta-crystallins and of gamma-crystallins are largely insoluble. 8. The brown reaction products of glutathione or cysteine with 1,2-naphthaquinone catalyse the oxidation of ascorbic acid in the same way as 1,2-naphthaquinone itself. 9. These results are discussed in relation to naphthalene-induced cataract.

scholarly journals The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, peptides and proteins

1968 ◽  
Vol 108 (3) ◽  
pp. 383-391 ◽  
Author(s):  
R. B. Freedman ◽  
G. K. Radda
Keyword(s):  
Amino Acids ◽  
Glutamate Dehydrogenase ◽  
Free Amino ◽  
Reactive Species ◽  
Second Order ◽  
Amino Group ◽  
Exponential Rate ◽  
Amino Groups ◽  
Sh Group ◽  
Kinetics Of

1. The kinetics of the reaction of 2,4,6-trinitrobenzenesulphonic acid with various amino acids, peptides and proteins were studied by spectrophotometry. 2. The reaction of the α- and ∈-amino groups in simple amino acids was found to be second-order, and the unprotonated amino group was shown to be the reactive species. 3. By allowing for the concentration of unreactive −NH3+ group, intrinsic reactivities for the free amino groups were derived and shown to be correlated with the basicities. 4. The SH group of N-acetylcysteine was found to be more reactive to 2,4,6-trinitrobenzenesulphonic acid than most amino groups. 5. The reactions of insulin, chymotrypsinogen and ribonuclease with 2,4,6-trinitrobenzenesulphonic acid were analysed in terms of three exponential rate curves, each referring to one or more amino groups of the proteins. 6. The reaction of lysozyme with 2,4,6-trinitrobenzenesulphonic acid was found to display an acceleration effect. 7. From the reaction of 2,4,6-trinitrobenzenesulphonic acid with glutamate dehydrogenase at several enzyme concentrations, it was possible to discern two sets of amino groups of different reactivity, and to show that the number of groups in each set was decreased by aggregation of the enzyme.

scholarly journals o-Quinones formed in plant extracts. Their reaction with bovine serum albumin

1969 ◽  
Vol 112 (5) ◽  
pp. 619-629 ◽  
Author(s):  
W. S. Pierpoint
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Plant Extracts ◽  
Bovine Serum ◽  
Succinic Anhydride ◽  
Thiol Group ◽  
Excess Oxygen ◽  
Amino Group ◽  
Amino Groups ◽  
Ellman's Reagent

1. The reactions between chlorogenoquinone, the o-quinone formed during the oxidation of chlorogenic acid, and bovine serum albumin depend on the ratio of reactants. 2. When the serum albumin is in excess, oxygen is not absorbed and the products are colourless. This reaction probably involves the thiol group of bovine serum albumin; it does not occur with bovine serum albumin which has been treated with p-chloromercuribenzoate, iodoacetamide or Ellman's reagent. 3. When bovine serum albumin reacts with excess of chlorogenoquinone, oxygen is absorbed and the products are red. The red colour is probably formed by reaction of the lysine ∈-amino groups of bovine serum albumin, as it is prevented by treating the protein with formaldehyde, succinic anhydride or O-methylisourea. 4. Bovine serum albumin modified by a 1·5-fold (BSA-Q) and a fivefold (BSA-Q2) excess of chlorogenoquinone were separated by chromatography on DEAE-Sephadex A-50, and some of their properties observed. 5. Reaction of BSA-Q2 with fluorodinitrobenzene suggests that the terminal α-amino group, as well as lysine ∈-amino groups, are combined with chlorogenoquinone.

scholarly journals The use of arginine analogues for investigating the functional organization of the arginine-binding site in lobster muscle arginine kinase. Role of the ‘essential’ thiol group

1980 ◽  
Vol 185 (3) ◽  
pp. 593-599 ◽  
Author(s):  
David C. Watts ◽  
Emmanuel O. Anosike ◽  
Barbara Moreland ◽  
R. J. Pollitt ◽  
C. R. Lee
Keyword(s):  
Amino Acids ◽  
Carboxy Group ◽  
Functional Organization ◽  
Arginine Kinase ◽  
Thiol Group ◽  
Difference Spectrum ◽  
Carbon Chain ◽  
Amino Groups ◽  
Difference Spectra ◽  
Nitrobenzoic Acid

1. The nature of arginine binding to lobster arginine kinase and the extent of its possible involvement with the ‘essential’ thiol group of the enzyme has been investigated with some inhibitory analogues of arginine. 2. Most of the analogues inhibit competitively, although mixed inhibition may occur if the α-carboxy group or α-amino group is absent. 3. The Ki values indicate that strength of binding depends on the length of the carbon chain (l-isoleucine>l-valine>l- α-aminobutyrate>l-alanine) and the integrity of the substituents on the α-carbon atom (l-arginine>agmatine and l-ornithine>putrescine). The guanidino group probably contributes little to substrate binding, but a positive charge near the δ-nitrogen atom appears to be important (l-ornithine>l -citrulline>l-α-aminobutyrate). A cyclic analogue, 2-carboxymethyl-3-oxo-2,3,5,6,7,8-hexahydro-1H-imidazo [1,2-a][1,3]diazepine-8-carboxylic acid, has a low Ki value similar to that of an equivalent straight-chain form, suggesting that arginine probably binds in a folded configuration. 4. The aliphatic l-amino acids give enzyme difference spectra similar to that with l-arginine and the integrity of the α-carboxy and α-amino groups appears to be a minimal but not sufficient requirement for this, as l-ornithine gives an atypical difference spectrum. A difference spectrum is interpreted as indicating an enzyme conformational change. No difference spectrum was observed with methylguanidine. 5. The ability of aliphatic α-l-amino acids to protect against inhibition by 5,5′-dithiobis-(2-nitrobenzoic acid) is proportional to the number of atoms in the carbon chain and inversely proportional to Ki. Ornithine gives greater protection than citrulline; analogues lacking the α-amino groups also protect. Agmatine, lacking the α-carboxy group, did not protect. 6. It is concluded that it is unlikely that the ‘essential’ thiol group in the enzyme interacts with any part of the arginine molecule during catalysis except, possibly, the α-carboxyl group.

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