Clostripain (Endoproteinase-Arg-C) is a two chain proteinase associated with collagenase and isolated from Clostridium histolyticum. It is highly specific for the carboxyl peptide bond of arginine. Clostripain has a sulfhydryl requirement; it is activated by dithiothreitol, cysteine, or other sulfhydryl containing reagents. The presence of calcium ions is essential. The enzyme is inhibited by oxidizing agents and sulfhydryl reactants and by Co²⁺, Cu²⁺, Cd²⁺, and heavy metal ions. Citrate, borate, and Tris anions are less inhibitory.
Clostripain is a cysteine-activated protease found, along with collagenase and other proteases, in culture filtrates of Clostridium histolyticum. It is unique in its specificity for the carboxyl peptide bond of arginine and its dependence on thiol and calcium ions.
Specificity
Clostripain selectively hydrolyzes arginyl bonds and lysyl bonds at a lower rate. It can also act as a transpeptidase with maximal activity at pH 7.6-9.0.
Composition
Clostripain is a heterodimer. The mature chain is composed of 526 residues. The two chains are held together by strong noncovalent forces. The catalytic sulfhydryl residue of the active site is believed to be Cys41 (heavy chain residue). The precursor contains a 27 amino acid putative signal peptide, a 23 amino acid propeptide, a 131 amino acid light chain subunit, a 9 amino acid linker peptide, and a 336 amino acid heavy chain subunit.
Molecular Characteristics
Both the heavy and light chains are encoded by a single gene with a 1581 nucleotide open reading frame (ORF). Upon expression of the gene, the entire ORF (the signal region, proregion, and 9 amino acid peptide linker) is transcribed. Postranslational processing produces the heterodimeric active enzyme.
Applications
Peptide mapping
Sequence analysis
Cell isolation
Hydrolysis/condensation of amide bonds
Peptide synthesis
Characteristics
Molecular Weight: 53.0 kDa (Theoretical); Light chain: 12.5 kDa, Heavy chain: 45 kDa
Optimal pH: 7.4-7.8 (activity against a-benzoyl-arginine ethyl ester)
Isoelectric point: 4.8-4.9
Extinction Coefficient: 87,890 cm⁻¹ M⁻¹ (Theoretical); E1%,280= 16.57 (Theoretical)
Active Site Residues: Cysteine (C41, heavy chain)
Activators: Sulfhydryl requirement: dithiothreitol, cysteine, or other reducing agents, Calcium ion is essential, Reducing agents
Inhibitors: EDTA, Oxidizing agents, Sulfhydryl reagents (such as TLCK), Co2+, Cu2+, Cd2+, and heavy metal ions, Citrate, borate and Tris anions partially inhibit
Assay
Method
The reaction velocity is measured as an increase in absorbance at 253 nm resulting from the hydrolysis of N-benzoyl-L-arginine ethyl ester. One unit hydrolyzes one micromole of BAEE per minute at 25°C and pH 7.6 under the conditions specified.
Reagents
0.075 M Sodium phosphate buffer, pH 7.6
7.5 mM Dithiothreitol (DTT)
0.75 mM N-Benzoyl-L-arginine ethyl ester (BAEE)
1.0 mM Calcium acetate containing 2.5 mM dithiothreitol (activation solution)
Enzyme
Dissolve or dilute the enzyme at a concentration of 1 mg/ml in water. Immediately prior to assay, dilute the enzyme further in 1.0 mM Calcium acetate containing 2.5 mM dithiothreitol to a concentration of 0.2-0.8 units/ml.
Procedure
Adjust spectrophotometer to 253 nm and 25°C.
Pipette into each cuvette as follows:
| 0.075 M phosphate buffer, pH 7.6 | 1.0 ml | | 7.5 mM DTT | 1.0 ml | | 0.75 mM BAEE | 1.0 ml |
|
Incubate in spectrophotometer for 3-5 minutes to achieve temperature equilibrium and establish blank rate, if any. At zero time, add 0.1 ml of appropriately diluted enzyme and record A253 for 4-5 minutes. Determine ΔA253/minute from the linear portion of the curve. Note: The reaction appears to be most linear with respect to enzyme concentration when ΔA253/min is between 0.007 and 0.030.
Calculation
where 1150 is the extinction coefficient of BAEE at 253 nm
