特异性
蛋白酶K具有广泛的底物特异性。即便存在洗涤剂的情况下，其仍可降解许多非变性状态的蛋白质。蛋白酶K分离自一种可在角质上生长的腐生真菌(Tritirachium album)。因此，蛋白酶K能够降解非变性状态的角质（头发），因而称为“蛋白酶K”[1]。其切割的主要位点为带有封闭氨基基团、邻近脂族或芳香族氨基酸羧基端的肽键。因其广泛的特异性，其较为常用[2,3,4]。
生理性质
蛋白酶K是稳定的S8家族丝氨酸碱性蛋白酶，在邻近活性位点组氨酸的位置含有两个二硫键和一个游离半胱氨酸[2,4]。
分子量：28,930 Da（氨基酸序列）[21]；28,500 Da (SDS-PAGE)[22]
pH范围：7.5-12.0（尿素变性后的血红素为底物），但多数情况下使用的pH范围为7.5-9.0[2,3]
温度曲线：最适活性温度37℃（20至60℃之间活性>最大活性的80%）[3]
pI: 8.92
消光系数：E1%=14.2(280 nm,10mM NaCl和5mM CaCl2，pH8.0[2]
活化剂：活化需要1-5mM Ca2+。去除酶中的钙离子（加入EDTA）后，会丢失25%的催化活性。但如果通过凝胶过滤去除EDTA-Ca2+复合物，则会总计丢失80%的酶活性，仅在向不含Ca2+的酶中加入过量的Ca2+时，才会发生少量活化[23]。
蛋白酶K在1% TRITON™ X-100之中激活，并在0.5% (w/v) SDS之中完全激活。SDS和尿素会使蛋白质底物变性，提高消解速率。在这些试剂作用下，蛋白酶K自身的消解速率要慢得多[3,19,20]。
单位定义：在pH7.5、37℃下，每分钟可水解尿素变性的血红素，产生1.0mmole (181mg) 酪氨酸所需的酶量为一单位。
抑制剂
蛋白酶K的抑制剂为DIFP或PMSF（后者使用的终浓度为5 mM）[3]。 EDTA只会使其部分失活，并不能起到抑制作用。蛋白酶K不会受到碘乙酸、胰蛋白酶特异性抑制剂TLCK、糜胰蛋白酶特异性抑制剂TPCK以及对-氯汞基苯甲酸盐抑制。
应用
*线粒体分离
*核酸纯化产物的蛋白质消解。在分子生物学应用中，蛋白酶K常用于消解无用的蛋白质，例如从微生物、培养细胞和植物的DNA或RNA制剂中消解核酸酶。[5-11]在核酸制剂之中，这种酶的使用浓度通常为50-200µg/ml，pH7.5-8.0，37℃。孵育时间30分钟至18小时不等。尽管在长期孵育时，蛋白酶K可以自动消解，但通常还是通过后续的本分萃取法使其变性[3]
*蛋白酶K已经用于去除阳离子蛋白质上结合的内毒素，如溶菌酶和核糖核酸酶A上的内毒素[12]
*确定酶在膜上的分布[13]
*处理石蜡包埋的组织，以便暴露抗原结合位点进行抗体标记[14]
*去除核酸酶进行原位杂交[15]
*传染性海绵状脑病(TSE)朊病毒研究及草拟的诊断测试方法利用蛋白酶K消解来自大脑组织样本的蛋白质[16,17]
*采用蛋白酶K消解法进行蛋白酶足迹实验，去除蛋白质-蛋白质表面互作[18]
制备说明
蛋白酶K可溶于水(1mg/ml)，获得无色透明的溶液。
溶解性和溶液稳定性
建议在-20℃下冻存粉末。产品可稳定保持至少2年。
蛋白酶K溶液在较广的pH范围内（4.0-12.5，最适pH8.0）保持稳定，同时在使用时可在25-65℃范围内保持稳定。pH8.0时，溶液至少可在4℃下稳定保存12个月[3]。pH4-11.5时，含有Ca2+ (1-6mM)的溶液预计可稳定保存数周。80%的硫酸铵悬液可在4℃下至少稳定保存12个月[2]。
产品列表
| 产品号 |
产品名称 |
规格 |
Cas |
标准包装 |
| P109033 |
蛋白酶K |
冻干粉，≥30 units/mg protein |
39450-01-6 |
25mg,100mg,500mg,1g,5g,25g,100g |
| P128666 |
蛋白酶K 来源于林伯氏白色念球菌 |
≥20 units/mg dry weight |
39450-01-6 |
25mg,100mg,1g |
| P301575 |
蛋白酶K 来源于林伯氏白色念球菌 |
≥500 units/mL, buffered aqueous glycerol solution |
39450-01-6 |
1ml,5ml,25ml |
阿拉丁是蛋白酶K大规模生产商。我们针对诊断生产和生物技术客户提供了定制配方制剂。详情可咨询官网www.aladdin-e.com。
文献列表
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[2]Ebeling, W., et al., Proteinase K from Tritirachium album Linder, Eur. J. Biochem., 47, 91 (1974).
[3]Enzymes of Molecular Biology, vol. 16, Burrell, M.M., ed. Humana Press (Totowa, NJ: 1993), p. 307. Kraus, E., and Femfert, U., Proteinase K from the Mold Tritirachium album Limber, Specificity and Mode of Action. Z. Physiol. Chem., 357, 937 (1976).
[4]Lizardi, P.M., and Engelberg, A., Rapid Isolation of RNA Using Proteinase K and Sodium Perchlorate. Anal. Biochem., 98, 116 (1979).
[5]Gross-Bellard, et al., Isolation of High Molecular Weight DNA from Mammalian Cells, Eur. J. Biochem., 36, 32-38 (1973).
[6]Molecular Cloning: A Laboratory Handbook, 2nd ed., Sambrook et al., eds., Cold Spring Harbor Press (Cold Spring Harbor, NY: 1989) p. 1.61 and p. B.16.
[7]Kasche, V., et al., A Two-step Procedure for Quantitative Isolation of Pure Double-strand DNA from Animal Tissues and Cell Cultures. Prep. Biochem., 11, 233 (1981).
[8]Hansen, J.N., Isolation of Higher Molecular Weight DNA from Bacillus cereus T Using Proteinase K. Prep. Biochem., 4, 473 (1974).
[9]Holm, C., et al., A Rapid, Efficient Method for Isolating DNA from Yeast. Gene, 42, 169 (1986).
[10]La Claire, J.W., and Herrin, D.L., Co-isolation of High-Quality DNA and RNA from Coenocytic Green Algae. Plant Mol. Biol. Reporter, 15, 263 (1997).
[11]Petsch, P., et al., Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the Limulus Amebocyte Assay: Application for Endotoxin removal from Cationic Proteins. Anal. Biochem., 259, 42 (1998).
[12]Brdiczyka, D., and Krebs, W., Localization of Enzymes by Means of Proteases. Biochem. Biophys. Acta, 297, 203 (1973).
[13]Short, B.G., et al., Automated Double Labeling of Proliferation and Apoptosis in Glutathione S-transferase-positive Hepatocytes in Rats. J. Histochemistry and Cytochemistry, 45, 1299 (1997).
[14]Angerer, L.M., et al., Identification of Tissue-Specific Gene Expression by in-situ Hybridization. Methods in Enzymology, 152, 649 (1987).
[15]Sakaguchi, S., et al., Accumulation of Proteinase K-Resistant Prion Protein (PrP) is Restricted by the Expression Level of Normal PrP in Mice Inoculated with a Mouse-Adapted strain of the Creutzfeldt-Jakob Disease Agent. J. Virology, 69, 7586 (1995).
[16]Bennion, B.J., and Daggett, V., Protein Conformation and Diagnostic Tests: the Prion Protein. Clinical Chemistry, 48, 2105 (2002).
[17]Hori, R., and Carey, M., Protease Footprinting Analysis of Ternary Complex Formation by Human TFIIA. J. Biol. Chem., 272, 1180 (1997).
[18]Hilz, H., et al., Stimulation of Proteinase K action by Denaturing Agents: Application to the Isolation of Nucleic Acids and the degradation of “Masked” Proteins. Eur. J. Biochem., 56, 103 (1975).
[19]Methods of Enzymatic Analysis, 3rd Edition, Bergmeyer, H.U., ed., Academic Press (New York, NY: 1983) vol. 2, p. 299.
[20]Jany, K.D., et al., Amino Acid Sequence of Proteinase K from the Mold, Tritirachium album Linder. Proteinase K; a Subtilisin-related Enzyme with Disulfide Bonds. FEBS Letters, 199, 139 (1986).
[21]Jany, K.D., and Mayer, B., Proteinase K from Tritirachium album linder, Molecular Mass and Sequence Around the Active Serine Residue. Biol. Chem. Hoppe-Seyler, 366, 485 (1985).
[22]Bajorath, J., et al., The Enzymatic Efficiency of Proteinase K is Controlled by Calcium. Eur. J. Biochem., 176, 441-447 (1988).
[23]IUBMB Enzyme Nomenclature: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/64.html