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A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe.

ABSTRACT: 1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.

SUBMITTER: Norris R

PROVIDER: S-EPMC1164111 | biostudies-other | 1976 Nov

REPOSITORIES: biostudies-other

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Project description:1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.

Project description:Serine proteases has been vastly reported to be found (and identified) only in seed extracts of Canavalia ensiformis (C. ensiformis), however, our group observed expressive serine protease activities in aqueous extracts from leaves, seeds, roots, and stem, especially in a leaf extract obtained with distillated water (CE-A). Additionally, this extract demonstrated very good stability in high temperatures and in the presence of chemical agents. In order to study the serine protease activity in leaves, CE-A was submitted to benzamidine-sepharose affinity column and an enriched fraction of serine proteases, named CE-ABza was purified 1.65 fold, yielding a total recovery of about 62%. Based upon zymographic analysis in a gelatin-SDS-PAGE-, the CE-ABza fraction presented an enzymatic activity band at approximately 90 kDa molecular mass region under non-reducing conditions and three bands at 17, 32 and also at 90 kDa under reducing condition. This fraction was able to hydrolyze, in a different way, peptidomimetic and protein substrates. The maximal activity was observed at basic pH values (8.0 to 9.5) and temperature about 40°C. These enzymes exhibited important thermal stability and were significantly inhibited by serine protease inhibitors, such as benzamidine and TPCK. Calcium, magnesium, manganese and zinc ions had a negative modulation on the CE-ABza activity. Mass spectrometry-based proteomics approach identified showed homology with 11 plant proteases from different legumes species, indicating their presence or demonstrating that CE-Bza proteases share specific region of the primary structure of theses enzyme, although the C. ensiformis genome and protein databases are yet to be publicly available. These results demonstrated that these molecules found in both the CE-A extract and the CE-ABza fraction presented important properties that could be used as potential pharmacological and biotechnological targets as therapeutic option. Besides, identification and characterization of C. ensiformis proteases contribute to the studies of plant enzymes.

Project description:Cysteine is unique among all protein-coding amino acids owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by both a broad range of redox mechanisms and various electrophiles derived from exogenous or endogenous sources. Measuring proteomic cysteines response to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling (ABPP) based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed as QTRP (Quantitative Thiol Reactivity Profiling), that relies on the ability of a commercially available thiol-reactive probe IPM to covalently label, enrich, and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g. control vs treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics, (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also outline here how to use QTRP for measuring the changes on proteomic cysteines upon redox perturbation and for identifying targeted cysteine sites of thiol-reactive electrophiles. We anticipate that this protocol should find broad applications in both redox/chemical biology and drug discovery. The protocol for sample preparation takes 3 days, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

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A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe.

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