The Structure of Pyruvate Carboxylase
D. B. Keech, J. C. Wallace in Pyruvate Carboxylase, 2018
As noted in Section III.G.1, all four subunits in chicken liver pyruvate carboxylase have an N-acetyl group on the amino-terminal residue(s) and are, therefore, not susceptible to sequence determination by the Edman degradation procedure. However, recent experiments by Khew et al.441 have shown that a brief exposure of the native enzyme to low concentrations of chymotrypsin achieves a rapid but selective cleavage of each subunit. When samples of this partial digest were analyzed by SDS-PAGE it was evident that the 112-kDa polypeptide chain had been almost quantitatively converted to a 75-kDa fragment plus smaller pieces. When this 75-kDa species was isolated and subjected to Edman degradation in the gasphase protein sequencer403 (Applied Biosystems, Inc.), an unambiguous N-terminal sequence of 23 residues could be deduced from an analysis of the PTHamino acid derivatives by reverse-phase HPLC. These results indicate that in all four subunits there are regions of high susceptibility to proteolysis, with cleavage in at least one of these, yielding 75-kDa fragments having identical N-terminal sequences for at least 23 residues.
Formation of Peptide Bonds — Proteases as “Activating Systems”
Willi Kullmann in Enzymatic Peptide Synthesis, 1987
When a given protein, peptide, or amino acid derivative fulfills the criteria characterizing a substrate of chymotrypsin, then its aromatic side-chain becomes anchored in the substrate binding cavity via a multitude of hydrophobic interactions. Beyond that, the mobility of the substrate is further restricted by the spatial constraints in the direct neighborhood of the binding area, by hydrogen bonds which attach the imino group and the carbonyl oxygen of the aromatic amino acid to the peptidic backbone of the enzyme, and by contacts of hydrophobic nature between the protease and the presumptive “leaving group”, R′, (cf. Figure 3) of the substrate. As a result, the substrate is fixed into a position that enables the enzyme to attack the reactive bond. As such the enzyme and substrate have been assembled to form the so-called Michaelis complex (Figure 3a).
A Novel Approach to Amino Acid Sequencing
Ajit S. Bhown in Protein/Peptide Sequence Analysis: Current Methodologies, 1988
Other enzymatic cleavages are also possible (Table 2). Chymotrypsin can be used under conditions similar to trypsin, and although the rates of cleavage for tryptophan, tyrosine, and phenylalanine are not the same — they occur in roughly that order — it should still be possible to sort them out. Tryptophan sites will be obvious because of the simultaneous appearance of bands in the NCS lane, and tyrosine and phenylalanine can be differentiated by prior treatment of a companion preparation with tetronitromethane to modify the tyrosines.26 Finally, it should be possible to identify glutamines by subjecting a preparation to partial deamidation under mildly basic conditions, followed by neutralization and digestion with staphylococcal protease.
Chymotrypsin attenuates adjuvant-induced arthritis by downregulating TLR4, NF-κB, MMP-1, TNF-α, IL-1β, and IL-6 expression in Sprague–Dawley rats
Published in Immunopharmacology and Immunotoxicology, 2022
Jianqiang Li, Linlin Wang, Guangting Zeng, Huilan Li, Jia Luo, Qijun Tian, Zanling Zhang
Therapy with proteolytic enzymes in rheumatic disorders has been used in previous studies [13]. Chintalacharuvu et al. [15] and Rovenská et al. [16] found the proteolytic enzyme drug Phlogenzym (bromelain, trypsin, rutin) had an inhibitory effect on collagen-induced arthritis in mice and rats, respectively. However, they did not explore the relevant mechanism further. Chymotrypsin is also a well-known proteolytic enzyme with anti-inflammatory effects [8]. The preliminary mechanism of chymotrypsin exerting an anti-rheumatic effect was studied in our research. We used CFA-induced rats as animal models in this study. Activated T cells could be detected, and multiple proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-17, and MMP were observed to participate in the pathological process of RA in an adjuvant-induced arthritis model [17].
Predicting Antibody Developability Profiles Through Early Stage Discovery Screening
Published in mAbs, 2020
Marc Bailly, Carl Mieczkowski, Veronica Juan, Essam Metwally, Daniela Tomazela, Jeanne Baker, Makiko Uchida, Ester Kofman, Fahimeh Raoufi, Soha Motlagh, Yao Yu, Jihea Park, Smita Raghava, John Welsh, Michael Rauscher, Gopalan Raghunathan, Mark Hsieh, Yi-Ling Chen, Hang Thu Nguyen, Nhung Nguyen, Dan Cipriano, Laurence Fayadat-Dilman
For peptide mapping by mass spectrometry, 100 μg of each sample was denatured with 30 µL of 8 M guanidine/1 M tris hydrochloride solution (15:1), reduced with 2 µL of 1 M dithiothreitol for 30 minutes at 60°C and alkylated with 5 µL of 1 M iodoacetamide for 45 minutes in dark. Before digestion, samples were buffer exchanged into 50 mM ammonium bicarbonate using 7 kDa molecular weight cut off ZEBA cartridges. Samples were digested with 2 µg of trypsin and chymotrypsin for 2 hours at 37°C. Digestion was quenched by the addition of 3 μL of 5 M hydrochloride to each sample. Data was acquired in a Dionex/QE plus MS using a linear gradient over 50 min from 2-36% acetonitrile in 0.1% formic acid. Samples were analyzed using PEAKS DB (Bioinformatics Solutions Inc.) for database searching as well as PepFinder (Thermo Fisher Scientific) and manual verification for the percent change assessment.
Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb
Published in mAbs, 2020
Wenkui Lan, Joseph J. Valente, Andrew Ilott, Naresh Chennamsetty, Zhihua Liu, Joseph M. Rizzo, Aaron P. Yamniuk, Difei Qiu, Holly M. Shackman, Mark S. Bolgar
The reduction of the disulfide bonds of the antibody was performed by mixing 1 mg of mAb-1 with 200 μL of 8 M guanidine hydrochloride, 18 μL of 0.2 M dithiothreitol, and 60 μL of 0.8 M trizma (pH = 7.6), then incubating the mixture at 37°C for 30 minutes. Alkylation of the cysteine was conducted by the addition of 18 µL of 0.4 M sodium iodoacetate to the reduced antibody and incubated in the dark for 15 minutes at room temperature. The desalting was followed by transferring the reduced and alkylated solution into a NAP-5 column, then exchanging with digestion buffer (50 mM, pH 7.6 Trizma, 10 mM CaCl2). Finally, the chymotryptic digestion was carried out by mixing desalted solution with chymotrypsin (protein: chymotrypsin ratio = 25:1) and incubating them at 37°C for 30 min.