特異性
蛋白酶K具有廣泛的底物特異性。即便存在洗滌劑的情況下，其仍可降解許多非變性狀態(tài)的蛋白質(zhì)。蛋白酶K分離自一種可在角質(zhì)上生長(zhǎng)的腐生真菌(Tritirachium album)。因此，蛋白酶K能夠降解非變性狀態(tài)的角質(zhì)（頭發(fā)），因而稱為“蛋白酶K”[1]。其切割的主要位點(diǎn)為帶有封閉氨基基團(tuán)、鄰近脂族或芳香族氨基酸羧基端的肽鍵。因其廣泛的特異性，其較為常用[2,3,4]。
生理性質(zhì)
蛋白酶K是穩(wěn)定的S8家族絲氨酸堿性蛋白酶，在鄰近活性位點(diǎn)組氨酸的位置含有兩個(gè)二硫鍵和一個(gè)游離半胱氨酸[2,4]。
分子量：28,930 Da（氨基酸序列）[21]；28,500 Da (SDS-PAGE)[22]
pH范圍：7.5-12.0（尿素變性后的血紅素為底物），但多數(shù)情況下使用的pH范圍為7.5-9.0[2,3]
溫度曲線：最適活性溫度37℃（20至60℃之間活性>最大活性的80%）[3]
pI: 8.92
消光系數(shù)：E1%=14.2(280 nm,10mM NaCl和5mM CaCl2，pH8.0[2]
活化劑：活化需要1-5mM Ca2+。去除酶中的鈣離子（加入EDTA）后，會(huì)丟失25%的催化活性。但如果通過凝膠過濾去除EDTA-Ca2+復(fù)合物，則會(huì)總計(jì)丟失80%的酶活性，僅在向不含Ca2+的酶中加入過量的Ca2+時(shí)，才會(huì)發(fā)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和尿素會(huì)使蛋白質(zhì)底物變性，提高消解速率。在這些試劑作用下，蛋白酶K自身的消解速率要慢得多[3,19,20]。
單位定義：在pH7.5、37℃下，每分鐘可水解尿素變性的血紅素，產(chǎn)生1.0mmole (181mg) 酪氨酸所需的酶量為一單位。
抑制劑
蛋白酶K的抑制劑為DIFP或PMSF（后者使用的終濃度為5 mM）[3]。 EDTA只會(huì)使其部分失活，并不能起到抑制作用。蛋白酶K不會(huì)受到碘乙酸、胰蛋白酶特異性抑制劑TLCK、糜胰蛋白酶特異性抑制劑TPCK以及對(duì)-氯汞基苯甲酸鹽抑制。
應(yīng)用
*線粒體分離
*核酸純化產(chǎn)物的蛋白質(zhì)消解。在分子生物學(xué)應(yīng)用中，蛋白酶K常用于消解無用的蛋白質(zhì)，例如從微生物、培養(yǎng)細(xì)胞和植物的DNA或RNA制劑中消解核酸酶。[5-11]在核酸制劑之中，這種酶的使用濃度通常為50-200µg/ml，pH7.5-8.0，37℃。孵育時(shí)間30分鐘至18小時(shí)不等。盡管在長(zhǎng)期孵育時(shí)，蛋白酶K可以自動(dòng)消解，但通常還是通過后續(xù)的本分萃取法使其變性[3]
*蛋白酶K已經(jīng)用于去除陽離子蛋白質(zhì)上結(jié)合的內(nèi)毒素，如溶菌酶和核糖核酸酶A上的內(nèi)毒素[12]
*確定酶在膜上的分布[13]
*處理石蠟包埋的組織，以便暴露抗原結(jié)合位點(diǎn)進(jìn)行抗體標(biāo)記[14]
*去除核酸酶進(jìn)行原位雜交[15]
*傳染性海綿狀腦病(TSE)朊病毒研究及草擬的診斷測(cè)試方法利用蛋白酶K消解來自大腦組織樣本的蛋白質(zhì)[16,17]
*采用蛋白酶K消解法進(jìn)行蛋白酶足跡實(shí)驗(yàn)，去除蛋白質(zhì)-蛋白質(zhì)表面互作[18]
制備說明
蛋白酶K可溶于水(1mg/ml)，獲得無色透明的溶液。
溶解性和溶液穩(wěn)定性
建議在-20℃下凍存粉末。產(chǎn)品可穩(wěn)定保持至少2年。
蛋白酶K溶液在較廣的pH范圍內(nèi)（4.0-12.5，最適pH8.0）保持穩(wěn)定，同時(shí)在使用時(shí)可在25-65℃范圍內(nèi)保持穩(wěn)定。pH8.0時(shí)，溶液至少可在4℃下穩(wěn)定保存12個(gè)月[3]。pH4-11.5時(shí)，含有Ca2+ (1-6mM)的溶液預(yù)計(jì)可穩(wěn)定保存數(shù)周。80%的硫酸銨懸液可在4℃下至少穩(wěn)定保存12個(gè)月[2]。
產(chǎn)品列表
| 產(chǎn)品號(hào) |
產(chǎn)品名稱 |
規(guī)格 |
Cas |
標(biāo)準(zhǔn)包裝 |
| P109033 |
蛋白酶K |
凍干粉，≥30 units/mg protein |
39450-01-6 |
25mg,100mg,500mg,1g,5g,25g,100g |
| P128666 |
蛋白酶K 來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 來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大規(guī)模生產(chǎn)商。我們針對(duì)診斷生產(chǎn)和生物技術(shù)客戶提供了定制配方制劑。詳情可咨詢官網(wǎng)www.aladdin-e.com。
文獻(xiàn)列表
[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