本产品为二次结晶，以水悬浮液形式提供。该制剂必须经稀释使酶溶解；稀释后的酶液使用前需经 0.22 μm 滤膜过滤。适用于 II 型肺细胞的分离。
储存条件：2‑8 ℃保存，禁止冷冻。
pH ≤ 3.5 条件下弹性蛋白酶不稳定。本产品在中性 pH、浓度＞0.25 % 时溶解性较差。建议先用氯化钾溶液或者碱性缓冲液配制母液，再稀释至反应体系或培养基中，需注意补偿由此带来的离子强度或 pH 变化。稳定 pH 范围：4.0‑10.4。
技术备注：1 个 SucAla3NA 单位约相当于 6 个弹性蛋白消化单位。水悬浮液需无菌操作，避免细菌污染。由于该水悬浮液具有粘稠特性，使用时需润洗样品瓶以充分回收瓶内物料。
猪胰弹性蛋白酶分子量为 25.9 kDa，最适 pH 8.5。该酶可水解多种蛋白底物；在各类蛋白酶中，其特殊之处在于能够水解天然弹性蛋白，而胰蛋白酶、糜蛋白酶、胃蛋白酶均无法降解该底物。大豆胰蛋白酶抑制剂、激肽释放酶抑制剂可抑制该酶的普通蛋白水解活性，但不抑制弹性蛋白水解活性。
弹性蛋白酶属于丝氨酸蛋白酶，亦可水解酰胺与酯类。该酶在胰腺内以无活性酶原形式合成，在十二指肠中被胰蛋白酶激活。
以下信息适用于猪源弹性蛋白酶。
底物特异性：
猪弹性蛋白酶 I 特异性切割 Ala‑Ala、Ala‑Gly 肽键；弹性蛋白酶 II 底物谱更广，偏好 P1 位为中‑大体积疏水氨基酸的底物（Gertler et al. 1977, Del Mar et al. 1980, Gestin et al. 1997）。猪源弹性蛋白酶活力最高，催化速率为人白细胞弹性蛋白酶的 20 倍（Bieth 1978, Bieth 1986, Largman 1983）。
水解反应分为多步：弹性蛋白酶与底物先形成吸附复合物；随后 Ser214 发生亲核进攻，生成酰基‑酶中间体，释放第一个产物（底物 C 端片段）；中间体经脱酰基步骤水解，再生出活性酶，同时释放第二个产物（Bieth 1986）。
分子组成：
催化三联体由三个通过氢键相连的氨基酸残基（H71、D119、S214）构成。多肽链包含两个反向平行 β‑桶结构域，二者围成容纳催化三联体的裂隙结构，同时含有少量 α‑螺旋（Bieth 2004）。
分子特征：
猪胰弹性蛋白酶为单条多肽链，共 240 个氨基酸，含 4 对二硫键（Sawyer et al. 1973）。与其他物种胰腺弹性蛋白酶序列同源性高，与大鼠同源性达 86%（MacDonald et al. 1982）。弹性蛋白酶 I、II 基因序列具有相似性，尤其在 5' 近端侧翼区，该区域包含 TATA 盒以及推定的组织特异性增强子序列（Ornitz et al. 1985, Stevenson et al. 1986, Swift et al. 1984, Tani et al. 1987, Gestin et al. 1997）。
应用：
1. 组织解离：弹性蛋白大量存在于结缔组织弹性纤维中，因此弹性蛋白酶常用于解离胞间纤维网络发达的组织；通常与胶原酶、胰蛋白酶、糜蛋白酶等其他酶复配使用。
2. 膜蛋白增溶
3. 蛋白质序列研究
弹性蛋白酶理化参数：
蛋白登录号：P00772
分子量：26.0 kDa
最适 pH：8.5
等电点：9.5
消光系数：54870 cm⁻¹・M⁻¹（理论）；E¹%₂₈₀ = 21.18（理论）
活性位点残基：组氨酸 H71、天冬氨酸 D119、丝氨酸 S214
抑制剂：
1. 可被丙氨酸、缬氨酸、亮氨酸、异亮氨酸的二肽衍生物竞争性抑制；
2. 天然来源抑制剂（如丝氨酸蛋白酶抑制剂 serpins）；
3. 改造型天然抑制剂；
4. 硫酸修饰脂质。
弹性蛋白酶活性检测
检测方法
改编自 Feinstein 等人 (1973)，并采用 Bieth 等人 (1974) 报道的高溶解性底物 N‑琥珀酰‑L‑Ala‑L‑Ala‑L‑Ala‑对硝基苯胺（Suc Ala₃NA）。底物水解会引起 410 nm 吸光度上升。
1 个酶活单位 (U) 定义：规定条件下，25 ℃、pH 8.0，每分钟水解 1 μmol Suc Ala₃NA 所需酶量。
试剂
1. 0.1 mol/L Tris 缓冲液，pH 8.0
2. 0.0044 mol/L Suc Ala₃NA 底物，溶于 Tris 缓冲液（2 mg/mL）
酶液
用 Tris 缓冲液配制成 1 mg/mL；临用前进一步稀释，使反应 ΔA/min 控制在 0.02‑0.04。
操作步骤
1. 分光光度计设置：波长 410 nm，温控 25 ℃。
2. 向比色皿中移取试剂：
Tris 缓冲液：2.7 mL
酶液：0.1 mL
3. 混匀，置于分光光度计内孵育 4‑5 min，完成温度平衡。
4. 向测试比色皿加入 0.2 mL 底物溶液，混匀；连续记录 3‑5 min 内 A₄₁₀吸光度变化。取曲线线性区间，计算 ΔA/min。
计算公式
比活(U/mg) = (ΔA₄₁₀/min) / [8.8 × 反应体系中酶质量浓度(mg/mL)]
This product is two‑times crystallized and supplied as an aqueous suspension. This preparation must be diluted to dissolve the enzyme. The diluted enzyme should be 0.22 micron filtered before use. Suitable for the isolation of Type II lung cells.
Store at 2-8°C. DO NOT FREEZE.
Stability/Storage: Elastase is unstable at pH ≤ 3.5. When stored as a dry powder the enzyme is stable for 6-12 months at 2-8°C. This product has poor solubility at neutral pH and at concentrations greater than 0.25%. It is helpful to make primary solutions in KCl or alkaline buffers before diluting into the reaction mixtures or media, compensating for ionic strength or pH changes. Stable at pH 4.0-10.4.
Technical Notes: 1 SucAla3NA unit is approximately equivalent to 6 elastin digestion units. Aqueous liquid suspensions should be aseptically handled to avoid bacterial contamination. Due to the viscous nature of the aqueous suspension the vial should be rinsed to recover contents.
Elastase is a serine protease that also hydrolyzes amides and esters. It is produced in the pancreas as an inactive zymogen, and activated in the duodenum by trypsin.
Porcine pancreatic elastase has a molecular weight of 25.9 kDa, and a pH optimum of 8.5. While elastase will hydrolyze a wide variety of protein substrates, it is unique among proteases in its ability to hydrolyze native elastin, a substrate not attacked by trypsin, chymotrypsin or pepsin. Soybean trypsin inhibitor and kallikrein inhibitor suppress proteolytic but not elastolytic activity.
Elastase is a serine protease that also hydrolyzes amides and esters. It is produced in the pancreas as an inactive zymogen, and activated in the duodenum by trypsin.
The following information applies to porcine elastase.
Specificity
Porcine elastase I is specific for Ala-Ala and Ala-Gly bonds, while elastase II has a broad specificity for substrates with medium to large hydrophobic amino acids in the P1 position (Gertler et al. 1977, Del Mar et al. 1980, and Gestin et al. 1997). Porcine elastase is the most potent elastase, having a rate 20-fold higher than that of human leukocyte elastase (Bieth 1978, Bieth 1986, and Largman 1983).
Hydrolysis occurs in several steps. An adsorption complex between elastase and its substrate is formed, followed by nucleophilic attack (S214) to form an acyl-enzyme intermediate, and release of the first product (the C-terminal end of the substrate). The intermediate is hydrolyzed in a deacylation step, regenerating the active enzyme and releasing the second product (Bieth 1986).
Composition
The catalytic triad is formed by three hydrogen-bonded amino acid residues (H71, D119, and S214). The polypeptide chain is composed of two antiparallel beta-barrel domains, which form a crevice containing the catalytic triad, and a small proportion of alpha-helices (Bieth 2004).
Molecular Characteristics
Porcine pancreatic elastase is composed of a single peptide chain of 240 amino acids, and contains 4 disulfide bridges (Sawyer et al. 1973). It has a high degree of sequence identity with pancreatic elastases from other species, such as rat with whom it shares 86% identity (MacDonald et al. 1982). Elastase I and II genes share sequence similarity, especially in the 5’ proximal flanking regions, which include the TATA box and a putative tissue-specific enhancer sequence (Ornitz et al. 1985, Stevenson et al. 1986, Swift et al. 1984, Tani et al. 1987, and Gestin et al. 1997).
Applications
Tissue dissociation: Because elastin is found in highest concentrations in the elastic fibers of connective tissues, elastase is frequently used to dissociate tissues that contain extensive intercellular fiber networks. For this purpose, it is usually used with other enzymes such as collagenase, trypsin, and chymotrypsin.
Membrane protein solubilization
Protein sequence studies
Characteristics of Elastase
Protein Accession Number: P00772
Molecular Weight: 26.0 kDa
Optimal pH: 8.5
Isoelectric point: 9.5
Extinction Coefficient: 54,870 cm⁻¹ M⁻¹ (Theoretical); E1%, 280 = 21.18 (Theoretical)
Active Site Residues: Histidine (H71), Aspartic acid (D119), Serine (S214)
Inhibitors:
Competitively inhibited by derivatives of dipeptides of alanine, valine, leucine, and isoleucine
Inhibitors from natural sources (e.g. serpins)
Modified natural inhibitors
Sulfate-modified lipids
Elastase Assay
Method
Derived from that of Feinstein et al. (1973) and using the more soluble substrate of Bieth et al. (1974). An increase in absorbance at 410 nm results from the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (Suc Ala3NA). One unit hydrolyzes one micromole of Suc Ala3NA per minute at 25°C and pH 8.0 under the specified conditions.
Reagents
0.1 M Tris buffer, pH 8.0
0.0044 M Suc Ala₃NA substrate dissolved in Tris buffer. (2 mg/ml)
Enzyme
Prepare a one mg/ml solution in Tris buffer. Immediately before use dilute further to obtain a rate of 0.02-0.04 ΔA/minute.
Procedure
Adjust the spectrophotometer to 410 nm and 25°C.
Pipette into each cuvette as follows:
Tris buffer 2.7 ml
Enzyme 0.1 ml
Mix and incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration. To test cuvette add 0.2 ml of substrate, mix, and record increase A410 for 3-5 minutes. Calculate ΔA/minute from the linear portion of the curve.
Calculation
Units/mg = (ΔA410/min) / (8.8 × mg enzyme / ml reaction mixture)
I2328404
危险品化学品经营许可证（带存储）