scholarly journals Purification and Characterization of Antibodies Directed against the α-Gal Epitope

BioChem ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 81-97
Author(s):  
Andreas Zappe ◽  
Julia Rosenlöcher ◽  
Guido Kohla ◽  
Stephan Hinderlich ◽  
Maria Kristina Parr
Keyword(s):  
Binding Affinities ◽  
Cell Surfaces ◽  
Purification And Characterization ◽  
Complex Type ◽  
Isoelectric Points ◽  
Human Sera ◽  
Affinity Resin ◽  
Glycan Profiling ◽  
2D Gel

The α-Gal epitope is an immunogen trisaccharide structure consisting of N-acetylglucosamine (GlcNAc)β1,4-galactose (Gal)α1,3-Gal. It is presented as part of complex-type glycans on glycoproteins or glycolipids on cell surfaces of non-primate mammalians. About 1% of all antibodies in human sera are specific toward α1,3-Gal and are therefore named as anti-α-Gal antibodies. This work comprises the purification and characterization of anti-α-Gal antibodies from human immunoglobulin G (IgG). A synthetically manufactured α Gal epitope affinity resin was used to enrich anti-α-Gal antibodies. Selectivity experiments with purified antibodies were carried out using enzyme-linked immunosorbent assays (ELISA), Western blotting, and erythrocyte agglutination. Furthermore, binding affinities toward α-Gal were determined by surface plasmon resonance (SPR) and the IgG distribution of anti α Gal antibodies (83% IgG2, 14% IgG1, 2% IgG3, 1% IgG4) was calculated applying ELISA and immunodiffusion. A range of isoelectric points from pH 6 to pH 8 was observed in 2D gel electrophoresis. Glycan profiling of anti α Gal antibodies revealed complex biantennary structures with high fucosylation grades (86%). Additionally, low amounts of bisecting GlcNAc (15%) and sialic acids (13%) were detected. The purification of anti-α-Gal antibodies from human IgG was successful, and their use as detection antibodies for α Gal-containing structures was evaluated.

scholarly journals Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

2018 ◽  
Vol 293 (41) ◽  
pp. 16057-16068 ◽  
Author(s):  
Bethany D. Harris ◽  
Jessica Schreiter ◽  
Marc Chevrier ◽  
Jarrat L. Jordan ◽  
Mark R. Walter
Keyword(s):  
Biological Activities ◽  
Human Interferon ◽  
Type I ◽  
Binding Affinities ◽  
Purification And Characterization ◽  
Cellular Assays ◽  
Mucosal Surfaces ◽  
Receptor Chain ◽  
The Common

IFNϵ and IFNκ are interferons that induce microbial immunity at mucosal surfaces and in the skin. They are members of the type-I interferon (IFN) family, which consists of 16 different IFNs, that all signal through the common IFNAR1/IFNAR2 receptor complex. Although IFNϵ and IFNκ have unique expression and functional properties, their biophysical properties have not been extensively studied. In this report, we describe the expression, purification, and characterization of recombinant human IFNϵ and IFNκ. In cellular assays, IFNϵ and IFNκ exhibit ∼1000-fold lower potency than IFNα2 and IFNω. The reduced potency of IFNϵ and IFNκ are consistent with their weak affinity for the IFNAR2 receptor chain. Despite reduced IFNAR2-binding affinities, IFNϵ and IFNκ exhibit affinities for the IFNAR1 chain that are similar to other IFN subtypes. As observed for cellular IFNAR2 receptor, the poxvirus antagonist, B18R, also exhibits reduced affinity for IFNϵ and IFNκ, relative to the other IFNs. Taken together, our data suggest IFNϵ and IFNκ are specialized IFNs that have evolved to weakly bind to the IFNAR2 chain, which allows innate protection of the mucosa and skin and limits neutralization of IFNϵ and IFNκ biological activities by viral IFN antagonists.

Separation, Partial Purification and Characterization of a Fatty Acid Hydroperoxide Cleaving Enzyme from Apple and Tomato Fruits

1982 ◽  
Vol 37 (3-4) ◽  
pp. 165-173 ◽  
Author(s):  
P. Schreier ◽  
G. Lorenz
Keyword(s):  
Fatty Acid ◽  
Inhibitory Effect ◽  
Fatty Acid Hydroperoxide ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isoelectric Points ◽  
Acid Hydroperoxide ◽  
Membrane Bound

Abstract A membrane-bound enzyme catalysing the cleavage of 13-hydroperoxy-(Z)-9,(E)-11-oc-tadecadienoic acid (13-LHPO) and 13-hydroperoxy-(Z)-9,(E)-11,(Z)-15-octadecadienoic acid (13-LnHPO) to C6-aldehydes was isolated and partially purified from apples and tomatoes. Attempts to employ Ultrogel AcA 34 and AcA 22 in a gel chromatographic purification step were partially frustrated by reaggregation phenomena. However, by using Sepharose CL-4 B an enzyme fraction (MW 200 000 Da) with lipoxygenase and fatty acid hydroperoxide cleaving activity could be separated from a high molecular-weight active eluate. By applying preparative isoelec­ tric focussing to the tomato protein we succeeded in separating the fatty acid cleaving activity from the lipoxygenase, because o f their different isoelectric points of pH 5.8 -6 .1 and pH 5.0, respectively, An 8.4-fold purification of the fatty acid cleaving activity was achieved. A pH-optimum of 5.5 and a Km-value of 2.6 × 10-5 м/1 for the 13-hydroperoxide of linoleic acid were measured. p-Chloromercuribenzoic acid (1 mм) showed significant inhibitory effect on the fatty acid hydroperoxide cleaving enzyme, but no evidence o f inhibition was found with 1 mм H2O2, KCN, DABCO and EDTA or superoxide dismutase (270 U). The maximum amount of fatty acid hydroperoxide decomposition (C6-aldehyde formation) was determined to be 59%.

Purification and Characterization of the Ro and La Antigens. Modulation of their Binding Affinities to Poly(U) by Phosphorylation and the Presence of ATP

1986 ◽  
Vol 367 (2) ◽  
pp. 671-680 ◽  
Author(s):  
Michael BACHMANN ◽  
Heinz C. SCHRÖDER ◽  
Karl G. WAGNER ◽  
Werner J. MAYET ◽  
Karin PFEIFER ◽  
...  
Keyword(s):  
Binding Affinities ◽  
Purification And Characterization

DNA Polymerase  s from Liver and Testes of Cherry Salmon, Oncorhynchus masou: Purification and Characterization of DNA Polymerase  s with Acidic Isoelectric Points

1996 ◽  
Vol 119 (1) ◽  
pp. 186-192 ◽  
Author(s):  
T. Yamaguchi ◽  
S. Nishimura ◽  
K. Takahashi ◽  
M. Yoshikuni ◽  
J. Masaki ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Oncorhynchus Masou ◽  
Purification And Characterization ◽  
Isoelectric Points

Purification and characterization of two iron superoxide dismutases ofPhytomonassp. isolated fromEuphorbia characias(plant trypanosomatids)

Parasitology ◽  
2004 ◽  
Vol 129 (1) ◽  
pp. 79-86 ◽  
Author(s):  
C. MARÍN ◽  
I. RODRÍGUEZ-GONZÁLEZ ◽  
A. B. HITOS ◽  
M. J. ROSALES ◽  
M. DOLLET ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Biochemical Marker ◽  
Subcellular Fractionation ◽  
Biochemical Properties ◽  
Superoxide Dismutases ◽  
Ionic Exchange ◽  
Purification And Characterization ◽  
Euphorbia Characias ◽  
Isoelectric Points

Two superoxide dismutases (SODI and SODII) have been purified by differential centrifugation, fractionation with ammonium sulphate followed by chromatographic separation (ionic exchange and affinity), from a plant trypanosomatid isolated fromEuphorbia characias, and then characterized for several biochemical properties. Both enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing superoxide dismutase. SODI had a molecular mass of approximately 66 kDa, whereas the molecular mass of SODII was approximately 22 kDa, both enzymes showing single bands. The isoelectric points of SODI and SODII were 6·8 and 3·6, respectively. The enzymatic stability persisted at least for 6 months when the sample was lyophilized and preserved at −80 °C. Digitonin titration and subcellular fractionation showed that both enzymes were in the cytoplasmic fraction, although part of SODII isoenzyme was also associated with glycosomes. We assayed these activities (SOD) in 18 trypanosomatid isolates on isoelectric focusing gels, and have demonstrated that the SOD is a biochemical marker sufficient to identify a trypanosomatid isolated from a plant as belonging to the genusPhytomonasand to distinguish between a truePhytomonasand other trypanosomatids that are capable of causing transient infections in plants.

scholarly journals Purification and characterization of eight glutathione S-transferase isoenzymes of hamster. Comparison of subunit composition of enzymes from liver, kidney, testis, pancreas and trachea

1992 ◽  
Vol 286 (2) ◽  
pp. 383-388 ◽  
Author(s):  
J J P Bogaards ◽  
B van Ommen ◽  
P J V van Bladeren
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Subunit Composition ◽  
Substrate Selectivity ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Amino Acid Sequence Analysis ◽  
Isoelectric Points ◽  
Made In

Eight dimeric isoenzymes of glutathione S-transferase (GST) were purified from liver, kidney and testis of the Syrian golden hamster, using S-hexylglutathione affinity chromatography and chromatofocusing. The isoenzymes were characterized according to their substrate selectivity, physical properties and amino acid sequence analysis. Thus a classification into Alpha, Mu and Pi classes was made in analogy with GSTs of other species. Two Alpha-class GSTs were purified, termed A1A1 (pI 8.9) and A1A2 (pI 8.6). Four Mu-class subunits were detected (M1-M4), all forming homodimers, with M2 and M3 also forming a heterodimer. The isoelectric points ranged from 5.9 to 8.6. One Pi-class isoenzyme was purified and termed P1P1 (pI 6.8). Using h.p.l.c. analysis, the subunit composition was determined in a number of organs. The major subunits in liver were A1 and M1. Subunit A1 was also the major subunit in the kidney. Subunit M1 was not detected in kidney, while subunit P1 was not found in the liver. Pancreas and trachea contained predominantly the Pi-class subunit, P1. GST in the testis was mostly of the Mu class. The major subunit was M4, and subunits M2 and M3 were exclusively detected in the testis.

Purification and Characterization of Galactosephilic Component Present on the Cell Surfaces ofStreptococcus sanguisATCC 10557

1983 ◽  
Vol 54 (3) ◽  
pp. 163-172 ◽  
Author(s):  
K. Nagata ◽  
M. Nakao ◽  
S. Shibata ◽  
S. Shizukuishi ◽  
R. Nakamura ◽  
...  
Keyword(s):  
Cell Surfaces ◽  
Purification And Characterization

Purification and characterization of two forms of Cu/Zn-superoxide dismutase from bovine erythrocytes by preparative isoelectric focusing

1981 ◽  
Vol 46 (9) ◽  
pp. 2140-2145 ◽  
Author(s):  
Hana Kovářová ◽  
Jan Šimša ◽  
Josef Křížala
Keyword(s):  
Superoxide Dismutase ◽  
Isoelectric Focusing ◽  
Visible Region ◽  
Alkaline Media ◽  
Ultraviolet Region ◽  
Purification And Characterization ◽  
Isoelectric Points ◽  
Relative Molecular Weight ◽  
Bovine Erythrocytes

Two forms of Cu/Zn-superoxide dismutase with isoelectric points (pI) 6.3 and 5.2 were isolated from bovine erythrocytes by preparative isoelectric focusing. Both forms show a relative molecular weight of 32 000 daltons corresponding to the value reported for the monomer molecule. From spectral analysis the maximum in the ultraviolet region of the spectrum (276 nm) is identical for both forms of superoxide dismutase whereas the maxima in the visible region are different (for the pI 5.2 form the maximum lies at 405 nm and for the pI 6.3 form at 695 nm). The different migration of the enzymatically active zones toward the anode during electrophoresis in alkaline media corresponds to their different isoelectric points.

scholarly journals Purification and characterization of two isoforms of Acanthamoeba profilin.

1986 ◽  
Vol 102 (1) ◽  
pp. 221-226 ◽  
Author(s):  
D A Kaiser ◽  
M Sato ◽  
R F Ebert ◽  
T D Pollard
Keyword(s):  
Molecular Weight ◽  
Actin Filaments ◽  
High Performance ◽  
Reversed Phase ◽  
Lag Phase ◽  
Purification And Characterization ◽  
Spontaneous Polymerization ◽  
Isoelectric Points ◽  
Pointed End

Acanthamoeba profilin purified according to E. Reichstein and E.D. Korn (1979, J. Biol. Chem. 254:6174-6179) consists of two isoforms (profilin-I and-II) with approximately the same molecular weight and reactivity to a monoclonal antibody but different isoelectric points and different mobilities on carboxymethyl-agarose chromatography and reversed-phase high-performance liquid chromatography. The isoelectric points of profilin-I is approximately 5.5 and that of profilin-II is greater than or equal to 9.0. Tryptic peptides from the two proteins are substantially different, which suggests that there are major differences in their sequences. At similar concentrations, both profilins prolong the lag phase at the outset of spontaneous polymerization and inhibit the extent of polymerization. Both forms also inhibit elongation weakly at the barbed end and strongly at the pointed end of actin filaments.

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