scholarly journals Synthesis and application of chemically reactive proteins by the reversible modification of protein amino groups with exo-cis-3,6-endo-epoxy-4,5-cis-epoxyhexahydrophthalic anhydride

1982 ◽  
Vol 203 (2) ◽  
pp. 345-350 ◽  
Author(s):  
R Schäfer
Keyword(s):  
Complete Recovery ◽  
Specific Protein ◽  
Enzymic Activity ◽  
Water Soluble ◽  
Organic Substrates ◽  
Amino Groups ◽  
Hydrophobic Membrane ◽  
Free Protein ◽  
Lysine Residues ◽  
Epoxy Groups

The reversible reaction of exo-cis-3,6-endo-epoxy-4,5-cis-epoxyhexahydrophthalic anhydride (EEHPA) with free protein amino groups is described. The free protein amino groups of lysozyme can be completely blocked through the reaction of the anhydride EEHPA. The chemically less reactive epoxy groups in EEHPA-modified lysozyme remain intact during modification of the protein and can be used for many subsequent chemical reactions. Hydrolysis of the modified inactive lysozyme at pH 2.5 results in deblocking and almost complete recovery of the enzymic activity of the protein. The epoxy groups in EEHPA-modified proteins have a great many potential uses: disaggregation of supramolecular structures, conversion of hydrophobic membrane proteins or tryptic peptides into water-soluble coloured proteins or peptides, inhibition of tryptic cleavage at lysine residues, synthesis of chemically reactive proteins or enzymes for affinity chromatography or immobilized-enzyme technology, two-dimensional separation techniques for complex protein mixtures, detection of specific protein-binding sites for organic substrates or tumour diagnostics, synthesis of defined artificial glycoproteins for biophysical and cytochemical studies and chemical synthesis of radioactively labelled proteins.

scholarly journals Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem II Membranes

2005 ◽  
Vol 37 (11) ◽  
pp. 737-742 ◽  
Author(s):  
Jin-Peng Gao ◽  
Zhen-Hua Yong ◽  
Feng Zhang ◽  
Kang-Cheng Ruan ◽  
Chun-He Xu ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Methyl Esters ◽  
Binding Activity ◽  
Water Soluble ◽  
Carboxyl Groups ◽  
Amino Groups ◽  
Charged Amino Acids ◽  
Lysine Residues ◽  
Glycine Methyl Ester ◽  
Positively Charged

Abstract To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with Nsuccinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4–10 and 10–14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3–4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.

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.

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.

scholarly journals Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies

1982 ◽  
Vol 205 (2) ◽  
pp. 361-372 ◽  
Author(s):  
E J Emanuel ◽  
A D Brampton ◽  
D R Burton ◽  
R A Dwek
Keyword(s):  
Immunoglobulin G ◽  
Water Soluble ◽  
Small Peptide ◽  
Experimental Conditions ◽  
Salt Ions ◽  
Salt Range ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Equilibrium Process ◽  
Glycine Ethyl Ester

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q-immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15-0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9-12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338-344].

A simple procedure using trinitrobenzenesulphonic acid for monitoring proteolysis in cheese

1988 ◽  
Vol 55 (4) ◽  
pp. 585-596 ◽  
Author(s):  
Anna Polychroniadou
Keyword(s):  
Simple Procedure ◽  
Water Soluble ◽  
Acid Solutions ◽  
Amino Groups ◽  
Total N ◽  
Cheese Ripening ◽  
Amino Acid Solutions ◽  
Hydrolysis Of ◽  
Good Agreement

SummaryA simple, rapid and sensitive spectrophotometric assay was developed and evaluated for monitoring proteolysis during cheese ripening, based on the fact that α-amino groups released by hydrolysis of cheese proteins react with trinitrobenzenesulphonic acid to form products that absorb strongly at 420 nm. A linear relationship was shown to exist between A420 and concentration of free α amino groups up to 0·5 HIM (r = 0·999, 38 df, P < 0·001). Repeatability of the method was satisfactory. The coefficient of variance was 0·53% for amino acid solutions and 1·19% for cheese extracts. Average recovery of glycine added to the cheese was 104 ± 2·9%. A comparison of the above method with that of determination of water-soluble N to total N ratio showed that there was good agreement between these two methods of assessment of proteolysis in cheese (r = 0·857, 32 df, P < 0·001). Mainly Feta and Teleme cheese were examined, but a similar correlation was obtained with hard Greek cheeses. Analytical conditions of the procedure are discussed.

Visible photostability of some ruthenium and platinum phthalocyanines in water and in the presence of organic substrates

2010 ◽  
Vol 14 (06) ◽  
pp. 499-508 ◽  
Author(s):  
Manuela Carchesio ◽  
Lucia Tonucci ◽  
Nicola d'Alessandro ◽  
Antonino Morvillo ◽  
Piero Del Boccio ◽  
...  
Keyword(s):  
Visible Light ◽  
Visible Light Irradiation ◽  
Carbonyl Compounds ◽  
Furfuryl Alcohol ◽  
Phenylacetic Acid ◽  
Water Soluble ◽  
Light Irradiation ◽  
Organic Substrates ◽  
Visible Spectra ◽  
Trapping Agent

Water-soluble, metal-tetrasubstituted phthalocyanines ( -SO3H , MPcS and -COOH , MPcC) of platinum and ruthenium were synthesized and their photostability to visible light irradiation was determined. For the ruthenium phthalocyanines, the characteristic visible Q band of the phthalocyanines was almost totally suppressed after five days of irradiation. The platinum derivatives were instead more resistant to photodegradation, and the Q band did not decrease by more than 25%. The addition of carbonyl compounds to the phthalocyanine solution in water (at concentrations at least 1400-fold those of the phthalocyanines) dramatically accelerated the photobleaching of these phthalocyanine complexes. PtPcS turned from blue to green and to colorless with one day of visible-light irradiation in the presence of acetone. This effect decreased with the increase in molecular weight of the ketones (from acetone to 2-pentanone). The addition of alcohols (i.e. 1-butanol) or other organics (i.e. phenylacetic acid) did not affect the photostability of these metal-tetrasubstituted phthalocyanines. Dioxygen also had an important role, as when the solutions of phthalocyanines were carefully deaerated before irradiation, the visible spectra were preserved. The platinum phthalocyanines, as with the palladium analogs, sensitize the photoproduction of 1O2 , as shown by the formation of endoperoxide and its rearranged products in the presence of furfuryl alcohol (a singlet oxygen trapping agent).

scholarly journals Essential carboxy groups in xylanase A

1990 ◽  
Vol 270 (1) ◽  
pp. 91-96 ◽  
Author(s):  
M R Bray ◽  
A J Clarke
Keyword(s):  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Water Soluble ◽  
Significant Activity ◽  
Nitrobenzoic Acid ◽  
Woodward’S Reagent K ◽  
Activity Loss

An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.

scholarly journals Ecophysiological Distinctions of Haloarchaea from a Hypersaline Antarctic Lake as Determined by Metaproteomics

2016 ◽  
Vol 82 (11) ◽  
pp. 3165-3173 ◽  
Author(s):  
Bernhard Tschitschko ◽  
Timothy J. Williams ◽  
Michelle A. Allen ◽  
Ling Zhong ◽  
Mark J. Raftery ◽  
...  
Keyword(s):  
Low Complexity ◽  
Abundant Species ◽  
Specific Protein ◽  
Organic Substrates ◽  
Deep Lake ◽  
Nitrogen And Phosphorus ◽  
Content Type ◽  
Cold Environments ◽  
Vestfold Hills ◽  
Life On Earth

ABSTRACTDeep Lake in the Vestfold Hills is hypersaline and the coldest system in Antarctica known to support microbial growth (temperatures as low as −20°C). It represents a strong experimental model because the lake supports a low-complexity community of haloarchaea, with the three most abundant species totaling ∼72%. Moreover, the dominant haloarchaea are cultivatable, and their genomes are sequenced. Here we use metaproteomics linked to metagenome data and the genome sequences of the isolates to characterize the main pathways, trophic strategies, and interactions associated with resource utilization. The dominance of the most abundant member,Halohasta litchfieldiae, appears to be predicated on competitive utilization of substrates (e.g., starch, glycerol, and dihydroxyacetone) produced byDunaliella, the lake's primary producer, while also possessing diverse mechanisms for acquiring nitrogen and phosphorus. The second most abundant member, strain DL31, is proficient in degrading complex proteinaceous matter.Hht. litchfieldiaeand DL31 are inferred to release labile substrates that are utilized byHalorubrum lacusprofundi, the third most abundant haloarchaeon in Deep Lake. The study also linked genome variation to specific protein variants or distinct genetic capacities, thereby identifying strain-level variation indicative of specialization. Overall, metaproteomics revealed that rather than functional differences occurring at different lake depths or through size partitioning, the main lake genera possess major trophic distinctions, and phylotypes (e.g., strains ofHht. litchfieldiae) exhibit a more subtle level of specialization. This study highlights the extent to which the lake supports a relatively uniform distribution of taxa that collectively possess the genetic capacity to effectively exploit available nutrients throughout the lake.IMPORTANCELife on Earth has evolved to colonize a broad range of temperatures, but most of the biosphere (∼85%) exists at low temperatures (≤5°C). By performing unique roles in biogeochemical cycles, environmental microorganisms perform functions that are critical for the rest of life on Earth to survive. Cold environments therefore make a particularly important contribution to maintaining healthy, stable ecosystems. Here we describe the main physiological traits of the dominant microorganisms that inhabit Deep Lake in Antarctica, the coldest aquatic environment known to support life. The hypersaline system enables the growth of halophilic members of theArchaea: haloarchaea. By analyzing proteins of samples collected from the water column, we determined the functions that the haloarchaea were likely to perform. This study showed that the dominant haloarchaea possessed distinct lifestyles yet formed a uniform community throughout the lake that was collectively adept at using available light energy and diverse organic substrates for growth.

Sign in / Sign up

Export Citation Format

Share Document