trypsin活测量方法.doc

Trypsin 背景介绍与实验设计Trypsin, introduction form sigma/life-science/metabolomics/enzyme-explorer/analytical-enzymes/trypsin.htmlPhysical Properties and In Vivo ProcessingTrypsin Enzyme Commission (EC) Number: Molecular Weight: 23.3 kDa1,2 (bovine & porcine)Extinction Coefficient: E1% = 12.9 - 15.4 (280 nm)3,4pI: 10.1 - 10.52,5(bovine)Trypsinogen Molecular Weight: 24 kDa5 (bovine)Extinction Coefficient: E1% = 14.4 (280 nm)pI: 9.32 (bovine)Trypsinogen, the proenzyme (zymogen) form of trypsin, is produced in the acinar exocrine cells of the pancreas. Three isoforms are excreted from the human pancreas. The cationic and anionic forms are the predominant human isoforms. The inhibitor-resistant mesotrypsinogen is found only in trace amounts.6 The proenzyme is activated only after it reaches the lumen of the small intestine. Enterokinase activates pancreatic trypsinogen to trypsin by the hydrolysis of a hexapeptide(for bovine trypsin at the Lys6 - Ile7 peptide bond) from the NH2 terminus. Bovine trypsinogen consists of a single polypeptide chain of 229 amino acids and is cross linked by six disulfide bridges. Trypsin can autocatalytically activate more trypsinogen to trypsin. Trypsin consists of a single chain polypeptide of 223 amino acid residues. This native form of trypsin is refered to as -trypsin. Autolysis of -trypsin (which is cleaved at Lys131- Ser132 in the bovine sequence) results in -trypsin which is held together by disulfide bridges. Trypsin is a member of the serine protease S1 family. The active site amino acid residues of trypsin include His46 and Ser183.2-5 Specificity, Kinetics, Substrates and Assay Methods Specificity and KineticsTrypsin will cleave peptides on the C-terminal side of lysine and arginine amino acid residues. The rate of hydrolysis is slower if an acidic residue is on either side of the cleavage site and no cleavage occurs if a proline residue is on the carboxyl side of the cleavage site. Trypsin will hydrolyze ester and amide linkages of several synthetic substrates: 2,7,8 The pH optimum of trypsin is 7 - 9.10 Assay Method11 The activity of most Sigma preparations is determined by a continuous rate spectrophotometric assay and expressed in BAEE units.Unit Definition: One BAEE unit will produce a A253 of 0.001 per min at pH 7.6 at 25 C using BAEE as substrate. Reaction volume = 3.2 mL (1 cm light path). Conditions: Temp = 25 C, pH = 7.6, A253nm, Light path = 1 cm In a 3.2 ml reaction mix, the final concentrations are 63 mM sodium phosphate, 0.23 mM N-benzoyl-L-arginine ethyl ester, 0.06 mM hydrochloric acid, and 100 units trypsin. Reagents:a. 67 mM Sodium Phosphate Buffer, pH 7.6 at 25 C (Prepare 100 ml in deionized water using Sodium Phosphate, Monobasic, Anhydrous, Sigma Prod. No. S0751. Adjust to pH 7.6 at 25 C with 1 M NaOH.) b. 0.25 mM Na-Benzoyl-L-Arginine Ethyl Ester Solution (BAEE) (Prepare 50 ml in Reagent A using N-Benzoyl-L-Arginine Ethyl Ester, Hydrochloride, Sigma Prod. No. B4500.) c. 1 mM Hydrochloric Acid Solution (HCl) (Prepare 50 ml in deionized water using concentrated Hydrochloric Acid, Sigma Prod. No. 258148.) d. Trypsin Enzyme Solution (Immediately before use, prepare a solution containing 500 BAEE units/ml of Trypsin in cold Reagent C.) Procedure:Pipette (in milliliters) the following reagents into suitable quartz cuvettes: Test Blank Reagent B (BAEE) 3.00 3.00 Equilibrate to 25 C. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add: Test Blank Reagent C (HCl) -0.20 Reagent D (Enzyme Solution) 0.20-Immediately mix by inversion and record the increase in A253nm for approximately 5 minutes. Obtain the A253nm/minute using the maximum linear rate for both the Test and Blank.Calculation:df = Dilution factor 0.001 = The change in A253nm/minute per unit of Trypsin at pH 7.6 at 25 C in a 3.2 ml reaction mix 0.20 = Volume (in milliliters) of enzyme usedNotes: 1. This assay procedure is not to be used to assay immobilized trypsins 2. For the USP/NF procedure refer to the USP monograph 3. This procedure is for informational purposes. For a current copy of Sigmas quality control procedure contact our Technical Service Department. Unit Conversions 1 BAEE M Unit = 200 BAEE Units 1 TAME M Unit = 0.27 BAEE M Units 1 BAEE M Unit = 3.64 TAME Units 1 TAME M Unit = 55 BAEE A253 Units 1 BAEE A253 Unit = 0.018 TAME M Unit 1 TAME M Unit = 180 TAME A247 Units 1 TAME A247 Unit = 0.33 BAEE Units 1 USP Unit = 3.0 BAEE Units 1 NF Unit = 1.1 USP Units Reconstitution and Solution Stability Trypsin solutions in 1 mM HCl (pH 3) are stable for approximately 1 year when aliquoted and stored at -20 C. The presence of Ca2+(20 mM) will also retard trypsins ability to selfdigest itself (autolysis) and will maintain the stability of the trypsin in solution.2,9 Trypsin retains most of its activity in 2.0 M urea, 2.0 M guanidine HCl, or 0.1% (w/v) SDS.13 Trypsin is reversibly denatured at high pH (above 11), by precipitation with TCA, or by high concentrations of urea (greater than 6.5 M).3 In order to abolish all trypsin activity, heating at 100 C in 1% (w/v) SDS for 5 minutes is required.14 Trypsinogen solutions are stable in acidic buffers (pH 2 - 4), while in neutral buffers the autocatalytic activation to trypsin occurs.References 1. Cunningham, L. W. Jr., Molecular Kinetic properties of crystalline diisopropyl phosphoryl trypsin. J. Biol. Chem., 211, 13-19 (1954). 2. Walsh, K. A., Trypsinogens and trypsins of various species. Meth. Enzymol., 19, 41-63 (1970). 3. Keil, B., in The Enzymes, 3rd ed., Vol. III, Boyer, P. D., Academic Press (New York, NY: 1971), pp. 250-275. 4. Shaw, E., et al., Evidence for an active center histidine in trypsin through use of a specific reagent, 1-chloro-3-tosylamido-7-amino-2-heptanone, the chloromethyl ketone derived from N-tosyl-L-lysine. Biochemistry, 4(10), 2219-2224 (1965). 5. Tietze, F., Molecular kinetic properties of crystalline trypsinogen. J. Biol. Chem., 204(1), 1-11 (1953). 6. Chen, J-M; Ferec, C., Genes, Cloned cDNAs, and Proteins of Human Trypsinogens and Pancreatitis-Associated Cationic Trypsinogen Mutations. Pancreas, 21, 57-62 (2000) 7. Burdon, R. H., et al., in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 9, 2nd ed., Allen, G., ed., Elsevier/North (New York, NY: 1989), pp. 73-104. 8. Enzyme Handbook, Vol. II, Barman, T. E., Springer-Verlag (New York, NY: 1969), pp. 618-619. 9. Enzyme Handbook, Vol. II, Barman, T. E., Springer-Verlag (New York, NY: 1969), p. 819. 10. Sipos, T., and Merkel, J. R., An effect of calcium ions on the activity, heat stability, and structure of trypsin. Biochemistry, 9(14), 2766-2775 (1970). 11. Bergmeyer, H.U., Gawehn, K., and Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed) Volume I, 2nd ed., 515-516, Academic Press, Inc.,New York, NY 12. Wang, S. S., and Carpenter, F. H., Kinetics of the tryptic hydrolysis of the oxidized B chain of bovine insulin. Biochemistry, 6(1), 215-224 (1967). 13. Carpenter, F. H., Treatment of trypsin with TPCK. Methods Enzymol., 11, 237 (1967). 14. Methods of Molecular Biology, Vol. 3, Smith, B. J., Humana Press, (New Jersey, 1988), pp. 57-69. Trypsin, Proteomics Grade from sigma/life-science/proteomics/mass-spectrometry/trypsin-proteomics-grade.html#Trypsin, Proteomics GradeTrypsin, Proteomics GradeSuperior Sequence Coverage Without Ambiguous Peaks!Proteomics Grade Trypsin (T6567) shows higher proteolytic efficiency than competitors products, generating more tryptic peptides leading to greater sequence coverage of your proteins of interest. Mass spectra are significantly simplified due to the reduced number of interfering autolytic peaks and their ambiguous adducts. (Concerns over the lack of internal calibrant peaks at low mass may be addressed by calibrating on matrix peaks that routinely appear in the mass spectrum under normal circumstances.)Proteomics Grade Trypsin has been extensively purified from porcine pancreas to enable accurate and precise cleavage on the carboxylic acid side of Arg and Lys residues. The enzyme has been exhaustively processed by reductive methylation to minimize autolysis and chymotryptic activity quenched by TPCK (N-Tosyl-L-phenylalanine chloromethyl ketone) treatment. Further purification steps including affinity chromatography and lyophilization from dilute acid produce a highly-purified, high specific activity trypsin purposely suited for the demanding criteria of proteomics research, and is designed to function for either in-solution or in-gel digests. The enzyme is conveniently packaged in five 20 g vials to ensure fresh enzyme is available for each use.Trypsin-ultra, Mass Spectrometry Grade (NEB)1 IVGGYTCAEN SVPYQVSLNA GYHFCGGSLI NDQWVVSAAH CYQYHIQVRL GEYNIDVLEG61 GEQFIDASKI IRHPKYSSWT LDNDILLIKL STPAVINARV STLLLPSACA SAGTECLISG121 WGNTLSSGVN YPDLLQCLVA PLLSHADCEA SYPGQITNNM ICAGFLEGGK DSCQGDSGGP181 VACNGQLQGI VSWGYGCAQK GKPGVYTKVC NYVDWIQETI AANSTrypsin-ultra, Mass Spectrometry Gradeiis a serine endopeptidase. It selectivelycleaves peptide bonds C-terminal to lysineand arginine residues (1). Trypsin-ultra istreated with L-(tosylamido-2-phenyl) ethylchloromethyl ketone (TPCK) to inactivate anyremaining chymotryptic activity. It is modifiedby acetylation of the -amino groups of lysineresidues to prevent autolysis. Trypsin-ultra (TPCKtreated)cleaves at Lys-Pro and Arg-Pro bondsat a much slower rate than other amino acidresidues (2).Product Source Isolated from bovine (Bos taurus) pancreasReaction Conditions 1X Trypsin-ultra, Reaction BufferIncubate at 37CTrypsin-ultra, Reaction Buffer:50 mM Tris-HCl20 mM CaCl2pH 8 25CUsage Concentration 100ng/lStorage Temperature -20C Molecular Weights Theoretical: 23675 daltons Specific Activity 2.1 mol/min/mg/cationic trypsin precursor Bos taurus(NEB为参考)/protein/2499861?report=genbank&log$=protalign&blast_rank=1&RID=KBS2F7KN016/nucleotide/829?report=genbank&log$=nucltop&blast_rank=1&RID=KBSZCEXR01R1 ATGCATCCCC TGCTTATCCT TGCCTTTGTG GGAGCTGCTG TGGCTTTCCC CTCGGACGAC61 GATGACAAGA TCGTCGGGGG CTACACCTGC GCAGAGAATT CCGTCCCTTA CCAGGTGTCC121 CTGAATGCTG GCTACCACTT CTGCGGGGGC TCCCTCATCA ATGACCAGTG GGTGGTGTCC181 GCGGCTCACT GCTACCAGTA CCACATCCAG GTGAGGCTGG GAGAATACAA CATTGATGTC241 TTGGAGGGTG GTGAGCAGTT CATCGATGCG TCCAAGATCA TCCGCCACCC CAAGTACAGC301 AGCTGGACTC TGGACAATGA CATCCTGCTG ATCAAACTCT CCACGCCTGC GGTCATCAAT361 GCCCGGGTGT CCACCTTGCT GCTGCCCAGT GCCTGTGCTT CCGCAGGCAC AGAGTGCCTC421 ATCTCCGGCT GGGGCAACAC CCTGAGCAGT GGCGTCAACT ACCCGGACCT GCTGCAATGC481 CTGGTGGCCC CGCTGCTGAG CCACGCCGAC TGTGAAGCCT CATACCCTGG ACAGATCACT541 AACAACATGA TCTGCGCTGG CTTCCTGGAA GGAGGCAAGG ATTCCTGCCA GGGTGACTCT601 GGCGGCCCTG TGGCTTGCAA CGGACAGCTC CAGGGCATTG TGTCCTGGGG CTACGGCTGT661 GCCCAGAAGG GCAAGCCTGG GGTCTACACC AAGGTCTGCA ACTACGTGGA CTGGATTCAG721 GAGACCATCG CCGCCAACAG CTGA本实验的实验方向：、实现表达和纯化；、固定化trypsin,开发可离心式的酶切工具去掉信号肽以后的表达蛋白序列1 MFPSDDDDKI VGGYTCAENS VPYQVSLNAG YHFCGGSLIN DQWVVSAAHC YQYHIQVRLG 61 EYNIDVLEGG EQFIDASKII RHPKYSSWTL DNDILLIKLS TPAVINARVS TLLLPSACAS 121 AGTECLISGW GNTLSSGVNY PDLLQCLVAP LLSHADCEAS YPGQITNNMI CAGFLEGGKD 181 SCQGDSGGPV ACNGQLQGIV SWGYGCAQKG KPGVYTKVCN YVDWIQETIA ANS*去掉信号肽以后的基因序列1 ATGTTCCCCT CGGACGACGA TGACAAGATC GTCGGGGGCT ACACCTGCGC AGAGAATTCC61 GTCCCTTACC AGGTGTCCCT GAATGCTGGC TACCACTTCT GCGGGGGCTC CCTCATCAAT121 GACCAGTGGG TGGTGTCCGC GGCTCACTGC TACCAGTACC ACATCCAGGT GAGGCTGGGA181 GAATACAACA TTGATGTCTT GGAGGGTGGT GAGCAGTTCA TCGATGCGTC CAAGATCATC241 CGCCACCCCA AGTACAGCAG CTGGACTCTG GACAATGACA TCCTGCTGAT CAAACTCTCC301 ACGCCTGCGG TCATCAATGC CCG