scholarly journals Effect of succinylation (3-carboxypropionylation) on the conformation and immunological activity of ovalbumin

1976 ◽  
Vol 155 (1) ◽  
pp. 171-180 ◽  
Author(s):  
S A Kidwai ◽  
A A Ansari ◽  
A Salahuddin
Keyword(s):  
Chemical Modification ◽  
Conformational Change ◽  
Intrinsic Viscosity ◽  
Antigenic Determinants ◽  
Amino Groups ◽  
Immunological Activity ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Stokes Radius ◽  
The Difference

The epsilon-amino groups of ovalbumin were modified with succinic anhydride; as many as 16 lysine residues were succinylated (3-carboxypropionylated). The five succinylated derivatives thus prepared were homogeneous with respect to the extent of chemical modification as shown by electrophoretic and immunological data. Succinylation of the amino groups altered electrophoretic mobility and isoionic pH of ovalbumin in the expected direction. U.v.-absorption and fluorescence spectra suggested changes in the microenvironment of the chromophores in the modified proteins. The difference-spectral results showed greater exposure of tyrosine and tryptophan residues in the succinylated ovalbumin. Increase in susceptibility to tryptic digestion, Stokes radius and intrinsic viscosity of native ovalbumin, which was observed on successive increase in the chemical modification, demonstrated a conformational change that was proportional to the extent of modification. The loss of immunological reactivity caused by chemical modification also indicated a conformational change in succinylated ovalbumin. The fact that the intrinsic viscosity of maximally modified ovalbumin was less than one-third of that for the completely denatured protein in 6M-guanidinium chloride suggested that the modified protein contained significant residual native structure. The latter presumably accommodates some antigenic determinants accounting for 37% residual immunological activity observed with maximally succinylated ovalbumin.

Letter: Specific Isotope Labeling for the Identification of Free N-Terminal Peptides of Proteins Separated by Polyacrylamide Gel Electrophoresis

2007 ◽  
Vol 13 (4) ◽  
pp. 307-309 ◽  
Author(s):  
Aniel Sanchez ◽  
Yassel Ramos ◽  
Yanni Solano ◽  
Luis Javier González ◽  
Lazaro Betancourt ◽  
...  
Keyword(s):  
Acetic Anhydride ◽  
Gel Electrophoresis ◽  
Isotope Labeling ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Amino Groups ◽  
Ion Distribution ◽  
Lysine Residues ◽  
Primary Amino ◽  
The Difference

We report here a method for the identification of free N-terminal peptides of in gel digested isolated proteins. It is based on the difference between the isotopic ion distribution of the N-terminal peptide and internal peptides. After guanidination of lysine residues, the primary amino groups of the gel-entrapped protein are blocked with an equimolar mixture of normal and deuterated acetic anhydride. Upon MS analysis, internal peptides display a normal isotopic ion distribution while the N-terminal peptide shows a complex isotopic ion distribution.

scholarly journals Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers

1988 ◽  
Vol 254 (2) ◽  
pp. 351-357 ◽  
Author(s):  
M S Shet ◽  
M Madaiah
Keyword(s):  
Acetic Anhydride ◽  
Chemical Modification ◽  
Succinic Anhydride ◽  
Structural Changes ◽  
Dicarboxylic Acids ◽  
Winged Bean ◽  
Amino Groups ◽  
Reductive Methylation ◽  
Tryptophan Residues ◽  
Haemagglutinating Activity

The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0.

Bovine Platelet Proteins III. Some Properties of Platelet Fibrinogen

1969 ◽  
Vol 21 (03) ◽  
pp. 428-440 ◽  
Author(s):  
N. O Solum ◽  
S Łopaciuk
Keyword(s):  
Intrinsic Viscosity ◽  
Rabbit Antiserum ◽  
Disc Electrophoresis ◽  
Carbohydrate Content ◽  
Plasma Fibrinogen ◽  
Total Amino Acid ◽  
Electrophoretic Mobilities ◽  
Bovine Plasma ◽  
Starch Gel ◽  
The Difference

Summary1. Some properties of purified bovine platelet fibrinogen have been described and the data compared to those obtained by parallel analysis of purified bovine plasma fibrinogen.2. A close similarity was found between platelet and plasma fibrinogen as to sedimentation coefficients, electrophoretic mobilities in starch gel and polyacrylamide disc electrophoresis, light absorption spectra in the range 240 mμ to 330 mμ, ability to form immunoprecipitate with a rabbit antiserum against bovine plasma fibrinogen, total amino acid composition and in N-terminal amino acids.Differences between the fibrinogens were found as to intrinsic viscosity, carbohydrate content and behaviour upon clotting by thrombin. Intrinsic viscosity in 0.3 M NaCl at 25° was 0.48 dl/g for platelet fibrinogen as compared to 0.26 dl/g for plasma fibrinogen. The carbohydrate content of platelet fibrinogen was 0.56 ± 0.10% 1.56±0.10% and 1.37±0.09% for sialic acid (calculated as N-glycolyl neuraminic acid), hexose (galactose/mannose 1:2) and hexosamine (glucosamine), respectively. These values were 6, 54 and 26% higher than those found for plasma fibrinogen. The difference in clotting behaviour indicated a slower polymerization rate of the fibrin monomers formed from platelet fibrinogen than of those formed from plasma fibrinogen.

The Action of Thrombin on Modified Fibrinogen

1983 ◽  
Vol 49 (03) ◽  
pp. 208-213
Author(s):  
A J Osbahr
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Lysine Residue ◽  
Fibrinopeptide A ◽  
Amino Groups ◽  
Lysine Residues ◽  
Citraconic Anhydride

SummaryThe modification of canine fibrinogen with citraconic anhydride modified the ε-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.

scholarly journals Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

1987 ◽  
Vol 105 (3) ◽  
pp. 1463-1469 ◽  
Author(s):  
C Fornieri ◽  
M Baccarani-Contri ◽  
D Quaglino ◽  
I Pasquali-Ronchetti
Keyword(s):  
Hydrophobic Interactions ◽  
Isonicotinic Acid ◽  
Lysyl Oxidase ◽  
Acid Hydrazide ◽  
Elastic Fibers ◽  
Amino Groups ◽  
Lysine Residues ◽  
Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

The interaction of bovine milk caseins with the detergent sodium dodecyl sulphate. II. The effect of detergent binding on spectral properties of caseins

1970 ◽  
Vol 37 (2) ◽  
pp. 259-267 ◽  
Author(s):  
G. C. Cheeseman ◽  
Dorothy J. Knight
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Difference Spectrum ◽  
Temperature Dependent ◽  
Difference Spectra ◽  
Negative Change ◽  
Tryptophan Residues ◽  
Hydrophobic Binding ◽  
The Difference

SummaryThe dissociation of casein aggregates by the detergent sodium dodecyl sulphate (SDS) gave rise to difference spectra and these spectra were characteristic for each of the different types of casein. Increase in absorption by the chromophore groups, tyrosine and tryptophan, when αs1- and β-casein aggregates were dissociated indicated binding of the detergent at regions of the molecule containing these residues. A decrease in absorption when κ-casein was dissociated indicated that the tyrosine and tryptophan residues were not in the region of the molecule to which the detergent was bound and that in the κ-casein aggregate these residues were in a more hydrophobic environment. Peaks on the difference spectra were obtained at 280 and 288 nm for αs1-casein and 284 and 291 nm for β-casein and troughs at 278 and 286 nm for κ-casein. The difference spectrum reached a maximum value when the αsl- and β-casein aggregates were dissociated and the further binding of SDS did not alter this value. The large negative change in the difference spectrum of κ-casein did not occur until after most of the aggregates were dissociated and did not reach a maximum until binding with SDS was complete. The value obtained for ΔOD was found to be temperature-dependent for β-casein-SDS interaction, but not for αs1- and κ-casein. Changes in spectra were also observed when αs1- and κ-casein interacted to form aggregates. The data obtained confirmed the importance of hydrophobic binding in casein aggregate formation and indicated the possible involvement of tyrosine and tryptophan residues in this binding.

Studies of Glutamate Dehydrogenase: Chemical Modification and Quantitative Determination of Tryptophan Residues

1974 ◽  
Vol 43 (2) ◽  
pp. 319-325 ◽  
Author(s):  
Veit Witzemann ◽  
Rudolf Koberstein ◽  
Horst Sund ◽  
Ihab Rasched ◽  
Hans Jornvall ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Quantitative Determination ◽  
Glutamate Dehydrogenase ◽  
Tryptophan Residues

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

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