分析酶

蛋白酶K

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真菌白色侧齿霉菌(Engyodontium album)(前称腐生真菌(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。24

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 °C(20至60 °C之间活性>最大活性的80%)3
pI: 8.92
消光系数:E1% = 14.2 (280 nm,10 mM NaCl和5 mM CaCl2,pH 8.0)2

活化剂:活化需要1-5 mM 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pH 7.5、37 °C下,每分钟可水解尿素变性的血红素,产生1.0 mmole (181 mg) 酪氨酸所需的酶量为一单位。

 

抑制剂

蛋白酶K的抑制剂为DIFPPMSF(后者使用的终浓度为5 mM)。3 EDTA(参见“活化剂”部分)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50-200 µg/ml,pH 7.5-8.0,37 °C。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(1 mg/ml),获得无色透明的溶液。

 

溶解性和溶液稳定性

Sigma建议在–20 °C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pH 8.0)保持稳定,同时在使用时可在25-65 °C范围内保持稳定。pH 8.0时,溶液至少可在4 °C下稳定保存12个月。3 pH 4-11.5时,含有Ca2+ (1-6 mM)的溶液预计可稳定保存数周。80%的硫酸铵悬液可在4 °C下至少稳定保存12个月。2

 

产品

货号
产品名称 加入购物车
P6556 Proteinase K from Tritirachium album lyophilized powder, ≥30 units/mg protein
P5056 Proteinase K from Tritirachium album ≥30 units/mg protein (biuret), lyophilized powder
P8044 Proteinase K from Tritirachium album 3-15 unit/mg solid, lyophilized powder
P5568 Proteinase K from Tritirachium album ≥500 units/mL, buffered aqueous glycerol solution
P2308 Proteinase K from Tritirachium album for molecular biology, >30 units/mg protein, lyophilized powder
P4850 Proteinase K from Tritirachium album for molecular biology, >800 units/mL, buffered aqueous glycerol solution, DNAse, Nickase and RNAse, none detected
SAE0009 Proteinase K from Tritirachium album, lyophilized powder, ≥30 units/mg protein

SRE0005 Proteinase K from Tritirachium album, Suitable for manufacturing of diagnostic kits and reagents

Sigma-Aldrich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。有关详情请联系当地SAFC代表。

 

参考文献

  1. Betzel, C., Three Dimensional Structure of Proteinase K at 0.15 nm Resolution. Eur. J. Biochem., 178, 155-171 (1988).
  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

 

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