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應用
胶原酶已用于动脉组织的制备,用于晚期糖基化终产物 (AGE) 的研究。这种酶还与其他蛋白酶一起用于人类肿瘤、小鼠肾、人脑、肺上皮和许多其他组织的解聚。也用于肝脏和肾脏灌注研究、胰腺的消化和非实质肝细胞的分离。检测浓度约为 1 mg/mL(每只大鼠肝脏总体积为 100 mL)时该酶对肝细胞的释放。
生化/生理作用
从组织中有效释放细胞需要胶原酶和中性蛋白酶的作用。每摩尔酶用 4 克原子钙 (Ca 2 + ) 激活胶原酶。培养滤液被认为含有至少 7 种不同的蛋白酶,分子量范围为 68-130 kDa。最佳 pH 值为 6.3-8.8。这种酶通常用于消化组织样本中的连接成分,以释放单个细胞。胶原酶处理可使部分细胞死亡。通常使用 0.1 至 5 mg/mL 的浓度进行消化。反应的持续时间从 15 分钟到数小时不等,可产生令人满意的细胞解离,且不会引起太多的细胞死亡。首选含钙和 BSA 的 Krebs Ringer 缓冲液,活性需要 Zn 2 + 。
每摩尔胶原蛋白酶被4g原子钙活化。 它被乙二醇-双(β-氨基乙基醚)-N,NN′,N′-四乙酸,β-巯基乙醇、谷胱甘肽、巯基乙酸和8-羟基喹啉抑制。
注意
供应时,本品在-20°C 下可稳定保存一年。在 37°C、50°C 和-20°C 条件下储存 30 天,FALGPA 或蛋白酶活性没有损失。如果快速冷冻 (10 mg/mL) 并在-20°C 下冷冻,粗胶原酶溶液是稳定的。进一步冻融循环将破坏溶液。置于冰上 7 小时后,产品仍保持 100% 活性。
單位定義
一个胶原蛋白消化单元(CDU)从牛跟腱胶原蛋白中释放的肽链量和1.0 μ亮氨酸,5小时,pH7.4,37℃,在钙离子存在下的茚三酮颜色相同。一个 FALGPA水解单元每分钟,25℃,水解1.0 μmole 呋喃基丙烯酰-Leu-Gly-Pro-Ala。一个中性蛋白酶单元水解酪蛋白,产生颜色和1.0 μmole 酪氨酸每5小时,pH7.5,37℃下产生的相同。一个梭菌蛋白酶单元在DTT存在下,pH7.6,25℃,每分钟水解1.0 μmole BAEE。
準備報告
溶液通常在 37°C 下用 TESCA 缓冲液(含 50 mM TES,0.36 mM 氯化钙,pH 7.4)制备,浓度为 1-2 mg/mL。
基底
訊號詞
Danger
危險分類
Eye Irrit. 2 - Resp. Sens. 1 - Skin Irrit. 2 - STOT SE 3
標靶器官
Respiratory system
儲存類別代碼
11 - Combustible Solids
水污染物質分類(WGK)
WGK 1
閃點(°F)
Not applicable
閃點(°C)
Not applicable
個人防護裝備
dust mask type N95 (US), Eyeshields, Faceshields, Gloves
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实验方案
To measure collagenase activity, N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala is used in a continuous spectrophotometric rate determination at 345 nm. Collagenase hydrolyzes collagen peptide bonds.
在测定胶原酶活性时,使用N-(3-[2-呋喃基]丙烯酰基)-Leu-Gly-Pro-Ala在345nm处进行连续分光光度法测定。胶原酶水解胶原肽键。
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