Collagenase I

The collagenase currently has approximately four types: Type I collagenase, Type II collagenase, Type III collagenase, and Type IV collagenase, which have specific applications: Type I Collagenase: Contains a relatively uniform variety of enzyme activities (including collagenase, caseinase, clostripain, and trypsin activity). It is commonly used for the preparation of epithelial cells and tissue cells. Type II Collagenase: Contains higher clostripain activity and is typically used for the preparation of cells from tissues such as heart, bone, muscle, thymus, and cartilage. Type III Collagenase: Contains lower protease activity and is frequently used for the preparation of mammary gland cells. Type IV Collagenase: Contains low trypsin activity and is usually employed for the preparation of pancreatic islet cells or for cell preparation experiments where receptor integrity needs to be maintained.

Collagenase I is classified as a proteinase derived from Bacillus histolyticus, having the capability to cleave the bonds between neutral amino acid and glycine residues within Pro-X-Gly-Pro sequences, which are prominently found in collagen proteins.

Collagenase I is relatively mild, with an initial balance of collagenase, caseinase, clostridium protease, and trypsin activity, and is well dissociated at physiological temperature and pH. The optimal pH of collagenase I is 7 ~ 8.

It has the unique ability to digest natural collagen and denatured collagen. Unique among proteases, collagenase I possesses the ability to break down the triple-helical natural collagen fibers that are abundant in connective tissues like skin, tendons, blood vessels, and bones. The proteolytic degradation facilitated by collagenase is employed in the enzymatic digestion of human tumors, mouse kidneys, adult and fetal brains, and various additional tissues, encompassing those featuring epithelial constituents. It acts exclusively on procollagen, breaking it and then being hydrolyzed by other proteases, without hydrolyzing fibrin and globulin. Detergents, hexachlorocyclohexane and heavy metal ions can reduce enzyme activity[1-5]. Collagenase I (Intravenous injection) could transiently reduceinterstitial fluid pressure (IFP) by digestion of collagen in tumors and increase tumor accumulation and gene expression of lipoplex[6].

Activity definition: One FALGPA hydrolysis unit hydrolyzes 1.0 µmole of furylacryloyl-Leu-Gly-Pro-Ala per min at pH 7.5 at 25 °C in the presence of calcium ions. One neutral protease unit hydrolyzes casein to produce color equivalent to 1.0 µmole tyrosine per 5 hr at pH 7.5 at 37℃. One clostripain unit hydrolyzes 1.0 µmole of BAEE per min at pH 7.6 at 25℃ in the presence of DTT.

References:
[1]. Pilcher BK, Sudbeck BD, et,al. Collagenase-1 and collagen in epidermal repair. Arch Dermatol Res. 1998 Jul;290 Suppl:S37-46. doi: 10.1007/pl00007452. PMID: 9710382.
[2]. Ohbayashi N, Yamagata N, et,al.Enhancement of the structural stability of full-length clostridial collagenase by calcium ions. Appl Environ Microbiol. 2012 Aug;78(16):5839-44. doi: 10.1128/AEM.00808-12. Epub 2012 Jun 8. PMID: 22685155; PMCID: PMC3406112.
[3]. Brandhorst H, Brandhorst D, et,al. Successful human islet isolation utilizing recombinant collagenase. Diabetes. 2003 May;52(5):1143-6. doi: 10.2337/diabetes.52.5.1143. PMID: 12716744.
[4]. Hamzeh Alipour, et al. Therapeutic applications of collagenase (metalloproteases): A review. Asian Pac J Trop Biomed, 2016, 6(11): 975-981.
[5]. O'Flanagan CH, Campbell KR, et,al. Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses. Genome Biol. 2019 Oct 17;20(1):210. doi: 10.1186/s13059-019-1830-0. PMID: 31623682; PMCID: PMC6796327.
[6]. Kato M, Hattori Y, Kubo M, Maitani Y. Collagenase-1 injection improved tumor distribution and gene expression of cationic lipoplex. Int J Pharm. 2012 Feb 28;423(2):428-34. doi: 10.1016/j.ijpharm.2011.12.015. Epub 2011 Dec 17. PMID: 22197775.

胶原酶目前大约有四种类型:I型胶原酶、II型胶原酶、III型胶原酶和IV型胶原酶,它们有特定的用途:I型胶原酶:含有相对统一的各种酶活性(包括胶原酶、酪蛋白酶、梭菌蛋白酶和胰蛋白酶活性)。通常用于上皮细胞和组织细胞的制备。II型胶原酶:含有较高的梭状蛋白酶活性,通常用于制备来自心脏、骨骼、肌肉、胸腺和软骨等组织的细胞。III型胶原酶:含有较低的蛋白酶活性,常用于乳腺细胞的制备。IV型胶原酶:含有低胰蛋白酶活性,通常用于胰岛细胞的制备或需要保持受体完整性的细胞制备实验。

胶原酶I是一种衍生自溶组织芽孢杆菌的蛋白酶,具有切割Pro-X-Gly-Pro序列中中性氨基酸和甘氨酸残基之间的键的能力,这种键在胶原蛋白中很常见。

胶原酶I相对温和,胶原酶、酪蛋白酶、梭状芽孢杆菌蛋白酶和胰蛋白酶活性初始平衡,在生理温度和pH下解离良好,最佳pH为7 ~ 8。

它具有独特的消化天然胶原蛋白和变性胶原蛋白的能力。胶原酶I在蛋白酶中是独一无二的,它具有分解存在于皮肤、肌腱、血管和骨骼等结缔组织中的三螺旋天然胶原纤维的能力。胶原酶促进的蛋白水解降解被用于人类肿瘤、小鼠肾脏、成人和胎儿大脑以及各种其他组织的酶消化,包括那些具有上皮成分的组织。它只作用于前胶原蛋白,破坏它,然后被其他蛋白酶水解,不水解纤维蛋白和球蛋白洗涤剂、六氯环己烷和重金属离子会降低酶的活性[1-5]。胶原酶-1(静脉注射)可通过消化肿瘤内的胶原而短暂降低间质液压力,增加肿瘤堆积和脂质体的基因表达[6]。

活性定义:1个FALGPA水解单元在钙离子存在下,在pH 7.5、25℃下每分钟水解1.0 µmol furylacryloyl-Leu-Gly-Pro-Ala。一个中性蛋白酶单位在37℃、pH 7.5条件下每5小时水解酪蛋白产生相当于1.0 µmol酪氨酸的颜色。在DTT存在下,一个clostripain单位在25℃、pH 7.6条件下每分钟水解1.0 µmol BAEE。