scholarly journals Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper

Separations ◽  
2021 ◽  
Vol 8 (5) ◽  
pp. 66
Author(s):  
Eleonora Pizzi ◽  
Trine Grønhaug Halvorsen ◽  
Christian J. Koehler ◽  
Léon Reubsaet
Keyword(s):  
Cytochrome C ◽  
Protein Binding ◽  
Covalent Binding ◽  
Protein Analysis ◽  
Blue Staining ◽  
Spectrometric Analysis ◽  
Trypsin Activity ◽  
Potassium Periodate ◽  
Immobilize Trypsin ◽  
Fundamental Insight

This paper describes smart sampling paper to be used for bottom-up protein analysis. Four different manners to immobilize trypsin on cellulose were evaluated. Untreated paper, potassium-periodate-functionalized paper (with and without post-immobilization reduction) and 2-hydroxyethyl methacrylate (HEMA)/2-vinyl-4,4-dimethylazlactone (VDM)-functionalized paper were all used to immobilize trypsin. For the evaluation, Coomassie Brilliant Blue staining of proteins on paper and the BAEE trypsin activity assay needed to be modified. These methods allowed, together with data from mass spectrometric analysis of cytochrome C digestions, us to acquire fundamental insight into protein binding, and trypsin action and activity on paper. All functionalized discs bind more protein than the untreated discs. Protein binding to functionalized discs is based on both adsorption and covalent binding. Trypsin immobilized on potassium-periodate-functionalized discs exhibits the highest trypsin activity when using cytochrome C as substrate. It is proven that it is trypsin attached to paper (and not desorbed trypsin) which is responsible for the enzyme activity. The use of discs on complex biological samples shows that all functionalized discs are able to digest diluted serum; for the best-performing disc, HEMA-VDM functionalized, up to 200 high-confidence proteins are qualified, showing its potential.

A preliminary proteomic survey of thein vitroexcretory/secretory products of fourth-stage larval and adultTeladorsagia circumcincta

Parasitology ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 535-543 ◽  
Author(s):  
H. CRAIG ◽  
J. M. WASTLING ◽  
D. P. KNOX
Keyword(s):  
Inflammatory Responses ◽  
Cysteine Proteinase ◽  
Nucleoside Diphosphate Kinase ◽  
Blue Staining ◽  
Spectrometric Analysis ◽  
Major Protein ◽  
Fourth Stage ◽  
Peptide Mass ◽  
Secretory Products

The nature of the proteins which comprise thein vitroexcretory/secretory products (ES) of the fourth-stage larva (L4) and adultTeladorsagia circumcinctaare largely undefined, despite the fact that this nematode induces profound changes, in part related to parasite ES, in the cellular architecture of the glands lining the abomasal surface of infected sheep and goats. In this study, the protein components of L4 and adult ES were fractionated using 1D gel electrophoresis and the major protein bands, detected by Coomassie blue staining, excised from the gel and subjected to tryptic digest and subsequent mass spectrometric analysis. The resultant peptide mass fingerprints were used to identify 15 L4 and 13 adult ES proteins. Several proteins, such as globin and some metabolic enzymes, were present in both ES. L4 ES alone contained thioredoxin peroxidase, an enzyme that can detoxify free radicals resulting from host inflammatory responses to the parasite, a cysteine proteinase which may aid penetration of the gastric mucosa and 2 different galectins which may influence cell differentiation and morphogenesis. Adult ES contained a nucleoside diphosphate kinase homologue, an enzyme which has been linked to cellular changes and can affect liquid secretion and goblet cell degranulation.

Activation of Platinum(IV) Prodrugs by Cytochrome c and Characterization of the Protein Binding Sites

2016 ◽  
Vol 13 (9) ◽  
pp. 3216-3223 ◽  
Author(s):  
Alessia Lasorsa ◽  
Olga Stuchlíková ◽  
Viktor Brabec ◽  
Giovanni Natile ◽  
Fabio Arnesano
Keyword(s):  
Cytochrome C ◽  
Protein Binding ◽  
Binding Sites ◽  
Protein Binding Sites

Identification of protein-derived tyrosyl radical in the reaction of cytochrome c and hydrogen peroxide: characterization by ESR spin-trapping, HPLC and MS

2002 ◽  
Vol 363 (2) ◽  
pp. 281-288 ◽  
Author(s):  
Steven Yue QIAN ◽  
Yeong-Renn CHEN ◽  
Leesa J. DETERDING ◽  
Yang C. FANN ◽  
Colin F. CHIGNELL ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Uv Irradiation ◽  
Phenyl Ring ◽  
Esr Spectra ◽  
Trapping Site ◽  
Spectrometric Analysis ◽  
Tyrosyl Radical ◽  
Radical Trapping ◽  
Radical Adduct ◽  
Hplc Peak

The reaction of cytochrome c and H2O2 is known to form a protein-centred radical that can be detected with the spin trap 2-methyl-2-nitrosopropane (MNP). To characterize the MNP/tyrosyl adduct structure that had previously been determined incorrectly [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498–15503], we eliminated unreasonable structure models by ESR studies with a series of 13C-labelled tyrosines, and photochemically synthesized an authentic MNP/tyrosyl adduct that has its trapping site on the C-3 position of the tyrosine phenyl ring. The observation of the identical ESR spectra for this radical adduct from the UV irradiation of 3-iodo-tyrosine and the adduct from the cytochrome c reaction demonstrated that the radical trapping site of MNP/tyrosyl is located on the equivalent C-3/C-5 positions instead of the C-1 position, as was proposed by Barr et al. In an on-line HPLC/ESR system, an identical retention time (17.7min) was observed for the ESR-active HPLC peak of the MNP/tyrosyl adduct from the following three reactions: (i) the tyrosine oxidation via horseradish peroxidase/H2O2; (ii) UV irradiation of 3-iodo-tyrosine and (iii) the reaction of cytochrome c with H2O2. This result demonstrated that the radical adducts of all three reactions are most probably the same. The mass spectrometric analysis of the HPLC fractions from reactions (i) and (ii) showed an ion at m/z 267 attributed to the MNP/tyrosyl adduct. We conclude that the cytochrome c-derived tyrosyl radical was trapped by MNP, leading to a persistent radical adduct at the C-3/C-5 positions of the tyrosine phenyl ring.

Effects of Specific Anion-Protein Binding on the Alkaline Transition of Cytochrome c

2001 ◽  
Vol 386 (1) ◽  
pp. 117-122 ◽  
Author(s):  
Gianantonio Battistuzzi ◽  
Marco Borsari ◽  
Antonio Ranieri ◽  
Marco Sola
Keyword(s):  
Cytochrome C ◽  
Protein Binding ◽  
Alkaline Transition

scholarly journals Haptenation: Chemical Reactivity and Protein Binding

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Itai Chipinda ◽  
Justin M. Hettick ◽  
Paul D. Siegel
Keyword(s):  
Protein Binding ◽  
Specific Binding ◽  
Chemical Reactivity ◽  
Covalent Binding ◽  
Kinetic Studies ◽  
Low Molecular Weight ◽  
Covalent Bonds ◽  
Protein Binding Sites ◽  
Disulfide Exchange ◽  
Histocompatibility Complex

Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

scholarly journals Covalent binding of arylazido derivatives of cytochrome c to cytochrome oxidase

FEBS Letters ◽  
1977 ◽  
Vol 81 (1) ◽  
pp. 147-150 ◽  
Author(s):  
R. Bisson ◽  
Heidi Gutweniger ◽  
C. Montecucco ◽  
R. Colonna ◽  
A. Zanotti ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Covalent Binding ◽  
Derivatives Of

Effect of different drugs on a cytochrome c – phospholipid bilayer membrane

1978 ◽  
Vol 56 (4) ◽  
pp. 555-563
Author(s):  
Michael A. Singer
Keyword(s):  
Cytochrome C ◽  
Protein Binding ◽  
Lipid Vesicles ◽  
Bilayer Membrane ◽  
Fatty Acyl ◽  
Phospholipid Bilayer ◽  
Butylated Hydroxytoluene ◽  
Na Permeability ◽  
Na Efflux ◽  
Temperature Interaction

Liposomes were prepared from dipalmitoyl phosphatidylcholine and dicetylphosphate and their interaction with the extrinsic membrane protein cytochrome c examined in terms of changes in 22Na permeability, electrophoretic mobility, protein binding, and motion of an incorporated spin label. The amount of cytochrome c bound displays no significant temperature dependence over the temperature range studied (from 30 to 55 °C) whereas cytochrome c causes an increase in 22Na efflux only above the phospholipid phase transition temperature. Interaction of the protein with the lipid vesicles causes no significant disturbance in the bilayer interior as monitored by the motion of the incorporated spin probe. The drugs 2,4-dinitrophenol and ethacrynic acid, both of which increase the magnitude of the vesicle negative charge, enhance both cytochrome c binding and its effect on 22Na permeability. In contrast, the local anesthetic dibucaine, which induces a positive surface charge on these liposomes, reduces both protein binding and the protein-induced increase in 22Na efflux. Finally, the chemicals butylated hydroxytoluene, 2-tert-butylphenol, and tert-butylbenzene, all of which cause early 'melting' of the phospholipid fatty acyl chains, block the capacity of cytochrome c to enhance 22Na permeability while having no effect on its binding to the vesicles.

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