Monoclonal Antibody Aggregation Associated with Free Radical Induced Oxidation

Oxidation is an important degradation pathway of protein drugs. The susceptibility to oxidation is a common concern for therapeutic proteins as it may impact product efficacy and patient safety. In this work, we used 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) as an oxidative stress reagent to evaluate the oxidation of therapeutic antibodies. In addition to the oxidation of methionine (Met) and tryptophan (Trp) residues, we also observed an increase of protein aggregation. Size-exclusion chromatography and multi-angle light scattering showed that the soluble aggregates induced by AAPH consist of dimer, tetramer, and higher-order aggregate species. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that inter-molecular disulfide bonds contributed to the protein aggregation. Furthermore, intrinsic fluorescence spectra suggested that dimerization of tyrosine (Tyr) residues could account for the non-reducible cross-links. An excipient screening study demonstrated that Trp, pyridoxine, or Tyr could effectively reduce protein aggregation due to oxidative stress. This work provides valuable insight into the mechanisms of oxidative-stress induced protein aggregation, as well as strategies to minimize such aggregate formation during the development and storage of therapeutic proteins.

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Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization

Journal of Bacteriology ◽

10.1128/jb.152.1.239-245.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 239-245

Author(s):

R M Berka ◽

M L Vasil

Keyword(s):

Pseudomonas Aeruginosa ◽

Phospholipase C ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Size Exclusion ◽

Tetradecyltrimethylammonium Bromide ◽

Heat Labile ◽

Hydrophobic Moiety ◽

Culture Supernatants

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.

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A Novel Degradation Pathway of Tissue Factor Pathway Inhibitor: Incorporation Into Fibrin Clot and Degradation by Thrombin

Blood ◽

10.1182/blood.v90.5.1883 ◽

1997 ◽

Vol 90 (5) ◽

pp. 1883-1892 ◽

Cited By ~ 28

Author(s):

Naoki Ohkura ◽

Kei-ichi Enjyoji ◽

Yu-ichi Kamikubo ◽

Hisao Kato

Keyword(s):

Tissue Factor ◽

Calcium Ion ◽

Tissue Factor Pathway Inhibitor ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Fibrin Clot ◽

Degradation Pathway ◽

Normal Plasma ◽

Tissue Factor Pathway ◽

Basic Region

Abstract Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF ) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from antithrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).

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Urease. X. A study by gel electrophoresis of its dissociation by sodium dodecyl sulfate

Canadian Journal of Biochemistry ◽

10.1139/o69-157 ◽

1969 ◽

Vol 47 (10) ◽

pp. 989-991 ◽

Cited By ~ 10

Author(s):

D. P. Blattler ◽

George Gorin

Keyword(s):

Sodium Dodecyl Sulfate ◽

Gel Electrophoresis ◽

Disulfide Bonds ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Dodecyl Sulfate ◽

Polypeptide Chains

Urease, m.w. 480 000, treated with an excess of sodium dodecyl sulfate is converted to a product of greatly increased mobility in polyacrylamide gel electrophoresis. We estimate its weight to be about 80 000. Treatment with excess thiol and detergent yielded the same product as detergent alone, indicating that the subunit or subunits do not contain polypeptide chains linked by disulfide bonds.

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Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00058-06 ◽

2006 ◽

Vol 13 (10) ◽

pp. 1155-1161 ◽

Cited By ~ 22

Author(s):

Donghee Cho ◽

Michael T. Collins

Keyword(s):

Solid Phase ◽

Expression Profiles ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Size Exclusion ◽

Enzyme Linked Immunosorbent Assay ◽

Infected Cattle ◽

Mycobacterium Paratuberculosis ◽

Serologic Tests ◽

Sds Page

ABSTRACT The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

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The kinetics of in vitro reoxidation and reduction of the inter heavy–light chain disulfide bond in an unusual murine immunoglobulin G myeloma protein lacking inter-heavy chain disulfide bonds

Canadian Journal of Biochemistry ◽

10.1139/o79-035 ◽

1979 ◽

Vol 57 (3) ◽

pp. 279-285 ◽

Cited By ~ 1

Author(s):

Maire E. Percy ◽

Lebe Chang ◽

Catherine Demoliou ◽

Reuben Baumal

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Peptide Mapping ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sedimentation Coefficient ◽

Disulfide Bridge ◽

Myeloma Protein ◽

Kinetics Of

After 5 years of subcutaneous transfer in Balb/C mice, our MOPC 173 myeloma tumour line (originally an IgG2a,κ H2L2-producer) exclusively synthesized an unusual IgG2b,κ protein lacking inter-heavy (H) chain disulfide bonds. This protein was designated MOPC 173B. On sodium dodecyl sulfate – polyacrylamide gel electrophoresis, it migrated with an apparent molecular weight of 77 000; following complete reduction and alkylation, the mobilities of its constituent H and light (L) chains were found to differ slightly from those of MOPC 173 H2L2. MOPC 173B was serologically identical to another typical IgG2b,κ myeloma protein, MOPC 195, and peptide mapping studies showed that it possessed only the inter H–L disulfide bond characteristic of typical IgG2b,κ proteins. In a nondissociating solvent, the sedimentation coefficient of the protein was 6.3S even at concentrations as low as 0.2 mg/ml, indicating that noncovalent interactions existed between two half-molecule subunits. Since this unusual IgG myeloma protein contained only a single category of interchain disulfide bridge, the inter H–L bond, it was an ideal model system for characterization of the kinetics of formation and reduction of interchain disulfide bonds. The kinetics of the glutathione-catalyzed reoxidation of the inter H–L disulfide bridge in MOPC 173B followed an apparent second-order rate equation. In contrast, reduction of its inter H–L bridge under anaerobic conditions with dithioerythritol in excess, was strictly a first-order process and not a simple reversal of the reoxidation. These studies provide the basis for the more complex mathematical models that describe the reoxidation and reduction of typical immunoglobulin molecules.

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A Novel and Potentially Multifaceted Dehydroascorbate Reductase Increasing the Antioxidant Systems is Induced by Beauvericin in Tomato

Antioxidants ◽

10.3390/antiox9050435 ◽

2020 ◽

Vol 9 (5) ◽

pp. 435 ◽

Cited By ~ 2

Author(s):

Martina Loi ◽

Silvana De Leonardis ◽

Giuseppina Mulè ◽

Antonio F. Logrieco ◽

Costantino Paciolla

Keyword(s):

Oxidative Stress ◽

Mass Spectrometry ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Calvin Cycle ◽

Mass Spectrometry Analysis ◽

Antioxidant Systems ◽

The Novel ◽

Main Band ◽

Chloroplast Mrna

Dehydroascorbate reductases (DHARs) are important enzymes that reconvert the dehydroascorbic acid (DHA) into ascorbic acid (ASC). They are involved in the plant response to oxidative stress, such as that induced by the mycotoxin beauvericin (BEA). Tomato plants were treated with 50 µM of BEA; the main antioxidant compounds and enzymes were evaluated. DHARs were analyzed in the presence of different electron donors by native and denaturing electrophoresis as well as by western blot and mass spectrometry to identify a novel induced protein with DHAR activity. Kinetic parameters for dehydroascorbate (DHA) and glutathione (GSH) were also determined. The novel DHAR was induced after BEA treatment. It was GSH-dependent and possessed lower affinity to DHA and GSH than the classical DHARs. Interestingly, the mass spectrometry analysis of the main band appearing on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a chloroplast sedoheptulose 1,7-bisphosphatase, a key enzyme of the Calvin cycle, and a chloroplast mRNA-binding protein, suggesting that the DHA reducing capacity could be a side activity or the novel DHAR could be part of a protein complex. These results shed new light on the ascorbate-glutathione regulation network under oxidative stress and may represent a new way to increase the plant antioxidant defense system, plant nutraceutical value, and the health benefits of plant consumption.

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Impact of Botrytis cinerea Contamination on the Characteristics and Foamability of Yeast Macromolecules Released during the Alcoholic Fermentation of a Model Grape Juice

Molecules ◽

10.3390/molecules25030472 ◽

2020 ◽

Vol 25 (3) ◽

pp. 472

Author(s):

Richard Marchal ◽

Thomas Salmon ◽

Ramon Gonzalez ◽

Belinda Kemp ◽

Céline Vrigneau ◽

...

Keyword(s):

Botrytis Cinerea ◽

Alcoholic Fermentation ◽

Periodic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Grape Juice ◽

Size Exclusion ◽

Periodic Acid Schiff ◽

Exclusion Chromatography ◽

The Impact

Botrytis cinerea is a fungal pathogen responsible for the decrease in foamability of sparkling wines. The proteolysis of must proteins originating from botrytized grapes is well known, but far less information is available concerning the effect of grape juice contamination by Botrytis. The impact from Botrytis on the biochemical and physico-chemical characteristics of proteins released from Saccharomyces during alcoholic fermentation remains elusive. To address this lack of knowledge, a model grape juice was inoculated with three enological yeasts with or without the Botrytis culture supernatant. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques (AgNO3 and periodic acid Schiff staining) was used in the study. When Botrytis enzymes were present, a significant degradation of the higher and medium MW molecules released by Saccharomyces was observed during alcoholic fermentation whilst the lower MW fraction increased. For the three yeast strains studied, the results clearly showed a strong decrease in the wine foamability when synthetic musts were inoculated with 5% (v/v) of Botrytis culture due to fungus proteases.

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Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1011 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1011-1018 ◽

Cited By ~ 13

Author(s):

M K Sauer ◽

D J Donoghue

Keyword(s):

Biological Activity ◽

Growth Factor ◽

Disulfide Bonds ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Sodium Dodecyl ◽

Biologically Active ◽

Platelet Derived Growth Factor ◽

Wild Type ◽

Type V

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.

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Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4438-4447.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4438-4447 ◽

Cited By ~ 36

Author(s):

Jung-Kul Lee ◽

Bong-Seong Koo ◽

Sang-Yong Kim ◽

Hyung-Hwan Hyun

Keyword(s):

Substrate Specificity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Catalytic Efficiency ◽

Industrial Applications ◽

Amino Acid Sequences ◽

Size Exclusion ◽

Mannitol Dehydrogenase ◽

High Substrate ◽

Candida Magnoliae

ABSTRACT Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k cat = 823 s−1, K m = 28.0 mM, and k cat /K m = 29.4 mM−1 s−1) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

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Identification of Polymorphic Outer Membrane Proteins of Chlamydia psittaci 6BC

Infection and Immunity ◽

10.1128/iai.69.4.2428-2434.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2428-2434 ◽

Cited By ~ 37

Author(s):

Regina J. Tanzer ◽

David Longbottom ◽

Thomas P. Hatch

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Disulfide Bonds ◽

Outer Membrane Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Late Phase ◽

Sodium Dodecyl ◽

Chlamydia Psittaci ◽

Developmental Cycle ◽

Lauryl Sarcosine

ABSTRACT The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.

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