梭菌蛋白酶（Endoproteinase-Arg-C）是一种与胶原酶相关的双链蛋白酶，分离自溶组织梭菌（Clostridium histolyticum）。该酶对精氨酸羧基端肽键具有高度特异性，其活性依赖于巯基化合物（如二硫苏糖醇、半胱氨酸或其他含巯基试剂）的激活；钙离子的存在对其功能至关重要。该酶可被氧化剂、巯基反应物、Co²⁺/Cu²⁺/Cd²⁺等重金属离子抑制，而柠檬酸根、硼酸根和Tris阴离子的抑制作用较弱。
作为溶组织梭菌培养滤液中的半胱氨酸激活蛋白酶，梭菌蛋白酶与胶原酶等蛋白酶共存，其独特之处在于兼具精氨酸羧基端肽键的特异性切割能力，以及对硫基化合物和钙离子的双重依赖性
特异性
梭菌蛋白酶以较低的速率选择性水解精氨酰键和赖氨酰键。它也能作为一种转肽酶，在pH 7.6-9.0时具有最大活性。
组成结构
梭菌蛋白酶是异二聚体。成熟链由526个残基组成。这两条链通过强大的非共价键结合在一起。活性位点的催化巯基残基被认为是Cys41（重链残基）。前体包含27个氨基酸的推定信号肽、23个氨基酸的前肽、131个氨基酸的轻链亚基、9个氨基酸的连接肽和336个氨基酸的重链亚基。
分子特征
重链和轻链都由具有1581个核苷酸的开放阅读框（ORF）的单个基因编码。基因表达后，整个ORF（信号区、前区和9个氨基酸肽接头）被转录。翻译后加工产生异二聚体活性酶。
应用
肽图分析
肽段序列分析
细胞分离
酰胺键水解/缩合反应
肽合成
特性
分子量: 53.0 kDa (理论值)；轻链：12.5 kDa，重链：45 kDa
最适pH：7.4-7.8 ( 对α-苯甲酰精氨酸乙酯的活性)
等电点：4.8-4.9
消光系数：87,890 cm⁻¹ M⁻¹（理论值）；E1%,280 = 16.57（理论值）
活性位点残基：半胱氨酸（C41，重链）
催化剂：巯基试剂需求：二硫苏糖醇、半胱氨酸或其他还原剂，钙离子为必需因子，还原剂
抑制剂：乙二胺四乙酸（EDTA）、氧化剂、巯基试剂（如甲苯磺酰赖氨酰氯甲酮/TLCK）、钴离子（Co²⁺）、铜离子（Cu²⁺）、镉离子（Cd²⁺）及其他重金属离子；柠檬酸根、硼酸根和Tris缓冲液阴离子可产生部分抑制
检测
方法
检测原理：通过监测 N-苯甲酰-L-精氨酸乙酯（BAEE） 水解反应在 253 nm 波长处 吸光度的上升速率测定酶活性。定义 1 个酶活单位（Unit） 为在 25°C、pH 7.6 的指定条件下，每分钟催化水解 1 微摩尔（μmol）BAEE 的能力。
试剂
0.075 M 磷酸钠缓冲液 , pH 7.6
7.5 mM 二硫苏糖醇 (DTT)
0.75 mM N-苯甲酰基-L-精氨酸乙酯 (BAEE)
1.0 mM 含有2.5 mM DTT的醋酸钙 (活化溶液)
酶
将酶溶于或稀释于水中，配制成1mg/ml的溶液。检测前立即用含2.5 mM二硫苏糖醇的1.0 mM醋酸钙溶液进行二次稀释，使酶浓度达到0.2-0.8 Unit/ml。
操作步骤：
将分光光度计波长调整为 253 nm，温度设定为 25°C；
按以下顺序向各比色皿中移液：
| 0.075 M 磷酸盐缓冲液, 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.
置于分光光度计中孵育3-5分钟以达到温度平衡，并建立空白反应速率（若需空白对照）。 在零点时，添加0.1 ml适当稀释的酶液，立即记录 253 nm波长处吸光度 A253 4-5分钟。根据反应曲线的线性区段计算ΔA253/min。 注：当ΔA253/min处于 0.007–0.030 区间时，酶浓度与反应线性相关性最佳。
计算
其中1150是BAEE在253nm的消光系数。
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
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