Proteinase K (Chimvuramabwe)
Katsi Nhamba: HC4502A
Proteinase K yakagadzikana serine protease ine yakafara substrate chaiyo.Iyo inosvibisa mapuroteni akawanda munharaunda yekuzvarwa kunyange pamberi pezvidziviriro.Humbowo kubva kukristaro uye mamorekuru magadzirirwo zvidzidzo zvinoratidza kuti enzyme ndeye subtilisin mhuri ine inoshanda saiti catalytic triad (Asp.39-Zvake69- Ser224)Nzvimbo huru ye cleavage ndiyo peptide bond iri padyo necarboxyl boka realiphatic uye anonhuwirira amino acids ane akavharika alpha amino mapoka.Inowanzoshandiswa kune yakafara yayospecificity.
Tsanangudzo
| Chitarisiko | Mvura isina ruvara kusvika kune yakatsvuka |
| Chiitiko | ≥800 U/ml |
| Protein concentration | ≥20 mg/ml |
| DNase | Hapana chaonekwa |
| RNase | Hapana chaonekwa |
Storage Conditions
Chengetedza kune tembiricha ye2-8 ℃.
Properties
| EC nhamba | 3.4.21.64 (Recombinant kubva kuTtirachium album) |
| Molecular uremu | 29 kDa (SDS-PEJI) |
| Isoelectric point | 7.81 |
| Optimum pH | 7.0-12.0 Fig.1 |
| Optimum tembiricha | 65 ℃ Fig.2 |
| pH kugadzikana | pH 4.5-12.5 (25℃, 16 h) Fig.3 |
| Thermal kugadzikana | Pasi pe50 ℃ (pH 8.0, 30 mins) Fig.4 |
| Activators | SDS, urea |
| Inhibitors | diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Applications
1. Genetic diagnostic kit
2. RNA uye DNA extraction kits
3. Kubviswa kwezvinhu zvisiri zveprotein kubva mumatishu, kuora kweprotein tsvina, seDNA vaccines uye kugadzirira kweheparin.
4. Kugadzirira kwechromosome DNA ne pulsed electrophoresis
5. Western blot
6. Enzymatic glycosylated albumin reagents in vitro diagnosis
Zvekungwaririra
Pfeka magirovhosi uye magirazi ekudzivirira paunenge uchishandisa kana kuyera, uye gara uine mhepo yakanaka mushure mekushandisa.Ichi chigadzirwa chinogona kukonzera ganda allergic reaction uye zvakanyanya kushatirwa kwemaziso.Kana inhaled, inogona kukonzera allergies kana asthma zviratidzo kana dyspnea.Inogona kukonzera kushatirwa kwekufema.
Assay
Tsanangudzo yeyuniti
Imwe unit (U) inotsanangurwa sehuwandu hwe enzyme inodiwa hydrolyze casein kugadzira 1 μmol tyrosine paminiti pasi pemamiriro anotevera.
Reagents kugadzirira
Reagent I: 1g mukaka casein yakanyungudutswa mu 50ml ye 0.1M sodium phosphate solution (pH 8.0), yakaiswa mu 65-70 ℃ mvura kwemaminetsi gumi nemashanu, yakamutswa uye yakanyungudutswa, yakanyoroveswa nemvura, inogadziriswa ne sodium hydroxide kusvika pH8.0, uye yakagadziriswa huwandu 100ml.
Reagent II: TCA solution:0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3mhinduro.
Reagent IV: Forint reagent yakanyungudutswa nemvura yakachena ka5.
Reagent V: Enzyme diluent: 0.1M sodium phosphate solution (pH 8.0).
Reagent VI: Tyrosine solution:0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine yakanyungudutswa ne0.2M HCl.
Maitiro
1. 0.5ml ye reagent I inofanodziya kusvika ku37℃, wedzera 0.5ml ye enzyme solution, sanganisa zvakanaka, uye incubate pa 37℃ kwe 10mins.
2. Wedzera 1ml ye reagent II kuti umise maitiro, sanganisa zvakanaka, uye ramba uchifukidzira 30mins.
3. Centrifugate reaction solution.
4. Tora 0.5ml supernatant, wedzera 2.5ml reagent III, 0.5ml reagent IV, sanganisa zvakanaka uye incuba pa 37℃ kwema30mins.
5. OD660yakatemwa seOD1;blank control group: 0.5ml reagent V inoshandiswa kutsiva enzyme mhinduro kuona OD660seOD2, DOD=OD1-OD2.
6. L-tyrosine yakajairika curve: 0.5mL yakasiyana-siyana L-tyrosine solution, 2.5mL Reagent III, 0.5mL Reagent IV mu 5mL centrifuge tube, incubate mu 37 ℃ kwe30mins, tsvaga OD660nokuda kwekusangana kwakasiyana kweL-tyrosine, ndokubva yawana chiyero chakajairwa Y=kX+b, apo Y ndiyo L-tyrosine concentration, X iri OD.600.
Calculation
2: Yese vhoriyamu yemhinduro mhinduro (mL)
0.5: Vhoriyamu ye enzyme solution (mL)
0.5: Reaction fluid volume inoshandiswa mukutsunga kwechromogenic (mL)
10: Nguva yekuita (min)
Df: Dilution yakawanda
CKuwedzera kwe enzyme (mg/mL)
References
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Commun.(1971);44:513.
3. Hilz, H.uye al.,Eur.J. Biochem.(1975);56:103–108.
4. Sambrook Jet al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989).
Figures
Fig. 1 Optimum pH
100mM buffer solution: pH6.0-8.0, Na-phosphate;pH8.0- 9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL
Fig. 2 Optimum temperatur
Reaction mu 20mM K-phosphate buffer pH 8.0.Enzyme concentration: 1mg/mL
Fig. 3 pH Kugadzikana
25℃,16 h-kurapa ne 50mM buffer mhinduro: pH 4.5-5.5, Acetate;pH 6.0-8.0, Na-phosphate;pH 8.0-9.0, Tris-HCl.pH 9.0-12.5, Glycine-NaOH.Enzyme concentration: 1mg/mL
Mufananidzo 4 Thermal kugadzikana
30 min-kurapwa ne50mM Tris-HCl buffer, pH 8.0.Enzyme concentration: 1mg/mL
Fig. 5 Kuchengeta stability at 25℃
