doc2.3 維持酵素活性Maintain activity
download 
Report Transcription
2.3 維持酵素活性Maintain activity
2 酵素活性測定法 Enzyme activity assay methods ● 2.1 催化反應 Catalytic reaction 活性測定時要注意一些基本原則 ● 2.2 酵素活性分析 Enzyme assay methods 反應速率 = 生成物 (P) / 時間 (t) ● 2.3 維持酵素活性 Maintain activity 很多酵素在細胞外容易失去活性 Juang RH (2005) EPA Use excess substrate [substrate] = 10 x Km - Substrate Enzyme Product might inhibit enzyme (feedback inhibition) Product NAD+ Remove product Measured directly NADH Changed to visible Enzyme catalysis and detection methods 耦合反應 ■ 酵 素 反 應 及 偵 測 方 法 Measurable? Coupling reaction Dehydrogenase Product transformed into measurable form Juang RH (2005) EPA ■ 基質量使用十倍 Km [Substrate] = 10 Km Velocity Vmax 1/2 Km 10 Km [substrate] Juang RH (2005) EPA ■ 酵素活性測定 Determine enzyme activity (SEPt) Optimized enzyme concentration Substrate excess 10 x Km E Product measurable pH t 酸鹼度 Proper reaction time P vo = t Temperature 溫度 Juang RH (2005) EPA ■ 反應速率需成線性 True velocity of the reaction Product True velocity False observation p p vo = t slope t Time t Juang RH (2005) EPA 2.2 酵素活性分析 Enzyme assay methods ● 2.2.1 酵素活性測定方法 Assay methods 在一定時間內測得生成物的產量 ● 2.2.2 中止酵素反應方法 Stop the reaction 中止酵素的方法不得破壞生成物或干擾測定 ● 2.2.3 連續測定法 Continuous measuring 連續測定可不用刻意中止酵素反應 ● 2.2.4 澱粉磷解脢活性分析 Assay for L-SP 以生化方法可以偵測到澱粉磷解脢的活性 Juang RH (2005) EPA 2.2.1 酵素活性測定方法 Assay methods ● a. 直接測定生成物 Measuring product directly 酒精去氫脢 (alcohol dehydrogenase, deHase) Alcohol ＋ NAD+ → Acetaldehyde ＋ NADH ＋ H+ ● b. 耦合反應法 Coupling reaction S → P → Q 可耦合到去氫脢反應 (coupled to deHase) ● c. 化學測定法 Chemical method ● d. 放射線測定法 Tracer method ● e. 測壓法 Manometry (for gaseous product) ● f. 電極 Electrode (for pH or O2 change) ● g. HPLC 檢定法 Your last choice Juang RH (2005) EPA ■ 輔脢 NADH 作用機制 Action of coenzyme NADH NADP+ Oxidized form NADPH Reduced form Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.86 H H H CONH2 CONH2 + N N P O P O Ribose Ribose H Adenine P O Adenine P O Ribose Ribose O O P P Juang RH (2005) EPA ■ Coenzyme NADH NAD+/NADH 的轉換可以 耦合 340 nm 吸光度變化 NAD+ NADH 340 nm NAD+ 吸 光 度 A 340 nm Dehydrogenase (去氫脢) Glyceraldehyde-3-P deHase ● Dehydrogenases use NADH or NADPH as coenzyme ● Have similar NAD+ Binding domain (Convergent evolution) NADH 240 280 320 360 400 Wave length (nm) Juang RH (2005) EPA Glyceraldehyde3-phosphate dehydrogenase NADH NADH Binding Domain (conserved) G-3-P substrate Binding Domain (variable) A typical dehydrogenase molecule contains two domains Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.25 ■ 一 個 典 型 的 去 氫 脢 ■ GUS activity assay - using synthetic substrate Substrate (colorless) COOH O O HO NO2 OH p-Nitrophenyl β-D-glucuronide (pNPG) COOH O OH + H2O GUS OH Products (yellow) HO OH + HO NO2 OH β-D-Glucuronic acid p-Nitrophenol (yellow) 415 nm GUS (glucuronidase) is extensively used for a reporter in cloning. Juang RH (2005) EPA ■ 酵 素 偵 測 方 法 Enzyme Measure directly Product Chemical method Enzyme detection methods Measured directly 耦合反應 NAD+ Tracer method Manometry Electrode HPLC 檢定法 Coupling reaction NADH Juang RH (2005) EPA ■ Coupled to dehydrogenase (NAD+-NADH) Hexokinase Glucose + ATP Glucose-6-P + ADP NAD+ Glc-6-P deHase NADH 6-P-Gluconic acid Juang RH (2005) EPA ■ 轉化脢 Invertase (IT) Sucrose Non-reducing sugar IT Glucose + Fructose Reducing sugars Reducing Power Juang RH (2005) EPA ■ 放射線測定法 Using radioactive tracer Glutathione γ-Glu-Cys-Gly N Artefact 假象 PCS X2 Phytochelatin γ-Glu-Cys-γ-Glu-Cys-Gly C Carboxypeptidase Y It is safer to measure phytochelatin directly by HPLC + Isolated for detection Juang RH (2005) EPA A sensor to monitor the quality of meat Enzyme electrode Oxygen electrode Oxygen consumption Hypoxanthine Hypoxanthine oxidase 牛頓 (1985) no. 23 p.108 2.2.4 澱粉磷解脢活性分析 Assay for L-SP Starch Phosphate + P n P Phosphorolysis P Starch-binding site Starch-binding site Catalytic site Catalytic site Starch biosynthesis + n-1 P Glc-1-P Juang RH (2005) EPA ■ Coupled to dehydrogenase Starch phosphorylase (phosphorolysis) Starch (n) + Pi Glc-1-P + Starch (n-1) Phosphoglucomutase Glc-6-P Glc-6-P deHase NAD+ NADH 6-P-Gluconic acid Juang RH (2005) EPA ■ 澱 粉 磷 解 活 性 分 析 及 干 擾 A B Activity staining Activity assay and interference Glc-1-P SP Ph Primer BA BA SP Native PAGE Glucose + Pi I2 Some other enzymes might interfere assay Direct observation of the color product Some other enzymes might lead to false (+) or (-) results Juang RH (2005) EPA 2.2.2 中止酵素反應方法 Stop enzyme reaction ● How to denature the enzyme effectively ? ● Change pH TCA Chemical Boiling Physical ● Rapidly heating ● Add denaturant SDS Chemical ● Add metal chelator EDTA Chemical ● Add enzyme inhibitor PMSF Chemical ● Add non-radioactive Physical substrate (pulse-chase) ● The product should not be destroyed ● No interference to the detection method Juang RH (2005) EPA Mathews & van Holde (1990) Biochemistry (1e) p.200, Fig. 6.25 ■ 蛋 白 質 的 四 級 構 造 1 Peptide bond Hydrogen bond 2 3 Disulfide bond Hydrophobic bond Hydrogen bond Four levers of protein structure 4 ■ SDS 在蛋白質表面均勻敷上一層負電 Native protein Protein is denatured to linear form SDS Hydrophobic core boiling Its surface covered with negatively charged SDS uniformly + Mercaptoethanol: to break the disulfide bonds Juang RH (2005) EPA Guanidine HCl NH2+ NH2- C -NH2 Mercaptoethanol HOCH2CH2SH SDS SO4- -(CH2)11CH3 Common denaturants for proteins Non-polar tail Juang RH (2005) EPA Polar head = Urea O NH2- C -NH2 = ■ 常 見 的 變 性 劑 = O -N H 2 NH 2-C H-bonds stabilize secondary structure Mathews et al (2000) Biochemistry (3e) p.164 β sheet O H2 N -C 2 H N α helix = ■ 蛋 白 質 二 級 構 造 ■ Pulse-chase Pulse Chase Pulse To measure the flow speed of water in a glass tube Juang RH (2005) EPA ■ 高爾基氏體的蛋白質運送 Golgi transportation Inner side Outer side Alberts et al (2002) Molecular Biology of the Cell (4e) p.736-737 2.3 維持酵素活性 Maintain enzyme activity ● 2.3.1 緩衝液 Buffer 可維持穩定的酸鹼度及離子濃度以保酵素活性 ● 2.3.2 試劑的保存 Reagents 試劑要依指示保存在適當的地方 ● 2.3.3 酵素活性之維持 Maintain the activity 注意酵素失活的原因有助保持其最高活性 ● 2.3.4 酵素活性單位 Enzyme activity unit Juang RH (2005) EPA ■ 弱酸鹼可作為緩衝分子 Buffer is weak acid/base Ka AH CH3COOH H-H equation Strong acid - A + H + - CH3COO - [A ] pH = pKa + log [AH] HCl - Cl + H + Juang RH (2005) EPA ■ 如 何 推 導 公 式 Ka = logKa = dissociated [A-] [H+] [AH] associated [A-] log [H+] [AH] logKa = log [H+] + [A-] log [AH] -log [H+] = -logKa + [A-] log [AH] pH = pKa + [A-] log [AH] (1) Ka 的定義 (2) 兩邊取 log 分解右邊 log (3) 移項 (4) 定義 -log 為 p Henderson-Hasselbalch equation How the pKa of a buffer contribute to its buffering effect How to derive H-H equation from Ka Juang RH (2005) EPA ■ 弱 酸 如 何 作 為 緩 衝 液 分 子 A- + H+ AH example Acetic acid CH3COOH = CH3COO- + H+ ◎ Ka is the dissociation constant Ka = [A-] [H+] pKa of acetic acid 1 = 10 5 (pKa = 5) [AH] ★ ◎ Ka is derived to get H-H equation 一、兩邊取 log 二、移項取出 [H+] 三、定義 p 為 -log (pH = -log[H+]) H-H equation pH = pKa + log 5 5 [A-] [AH] dissociated associated pH = constant pKa？ ….. when [A-] = [AH], log 1 = 0 Why weak acid could serve as a buffer ? Juang RH (2005) EPA ■ 弱酸在其 pKa 上下有緩衝作用 functional pH range pH Water pH pKa Acetic acid 5 ★ [OH-] H-H equation pH = pKa + log 6 5 Acetic acid has highest buffer effect at its pKa (pH 5) (just its pKa) [OH-] [A-] [AH] dissociated associated Juang RH (2005) EPA ■ 質子可以吸著或脫離一基團 Proton： The smallest and most abundant particle in the living cell controlling the pH and the charge property of a molecule lone pair electrons Amino H+ N H H+ N H H Carboxylic C H O H O O C O H+ Ampholyte: A molecule contains both positively and negatively charged groups Proton can enter or leave a functional group relatively freely Juang RH (2005) EPA ■ 胺基酸的緩衝作用範圍 Amino acid as a buffer pH 12 ★ 9 NH2 H+ 6 H- C- R Isoelectric point = COO- 3 pK2 ★ pK1 + pK2 2 pI pK1 0 Amino acid can be used as a buffer [OH] → Juang RH (2005) EPA ■ 各種常用緩衝液及其使用範圍 Common buffers Buffer pH Formate Citrate Acetate Phosphate HEPES Tris Borate Carbonate 3.0 - 4.5 3.0 - 6.2 3.7 - 5.5 5.8 - 8.0 6.5 - 8.5 7.1 - 8.9 9.1 - 9.0 9.7 - 10.7 Volatile, could be removed by lyophilization Universal 2 - 12 Contains several buffers at various pH ranges Remarks Bind with divalent metal ions Volatile, could be removed by lyophilization Precipitated with Ca; crystallized at low temperature Low toxicity, used in cell culture pH effected by temperature; special pH electrode required Bind with divalent metal ions Juang RH (2005) EPA ■ 緩衝液使用注意 Notice for two common buffers Diluted phosphate buffer raises its pH Tris is affected by temperature 9 pKa 8 Tris 7 Phosphate Tris Phosphate 7 0 8 10 20 30 Temperature 40 0 0.1 0.2 Ionic strength Juang RH (2005) EPA ■ 緩衝液常用的添加物 Some common additives Additives Action NaN3 (sodium azide) EDTA Concentration Antimicrobials Remove metal ions Antioxidant β-Mercaptoethanol Dithiothreitol (DTT or DTE) Antioxidant BSA (bovine serum albumin) Stabilizer 0.01% 0.1 - 1 mM 1 - 10 mM 1 - 5 mM 0.1 - 10 mg/mL Tween-20, Triton X-100 Glycerol, glucose PMSF, TPCK, TLCK, benzamidine etc. 0.5 - 0.05% 50% Surfactant Antifreeze Proteinase inhibitor Trace amount Juang RH (2005) EPA ■ Beta-mercaptoethanol α HOCH2CH2SH HOCH2CH2SH + o 1 2 β HOCH2CH3 HOCH2CH2S 2 HOCH2CH2S HOCH2CH2SH + H2O active oxidized form HOCH2CH2S Enzyme SH X Enzyme S Juang RH (2005) EPA ■ Dithiothreitol (DTT) CH2SH HO-C-H HO-C-H CH2SH H-C-OH HO-C-H CH2SH CH2SH Dithioerythritol (DTE) Dithiothreitol (DTT) 1 2 o2 CH2 H-C-OH S HO-C-H S CH2 Stable cyclic oxidized form Juang RH (2005) EPA ■ 緩衝液使用濃度範圍 Concentration ranges 1 mM 10 mM General buffers Enzyme assay 100 mM 1M Protein extraction Chromatography Juang RH (2004) ECX 2.3 維持酵素活性 Maintain enzyme activity ● 2.3.1 緩衝液 Buffer 可維持穩定的酸鹼度及離子濃度以保酵素活性 ● 2.3.2 試劑的保存 Reagents 試劑要依指示保存在適當的地方 ● 2.3.3 酵素活性之維持 Maintain the activity 注意酵素失活的原因有助保持其最高活性 ● 2.3.4 酵素活性單位 Enzyme activity unit Juang RH (2005) EPA ■ 試 劑 的 保 存 注 意 事 項 a. Avoid humidity Open the bottle of a frozen reagent only when its temperature has been brought back to the room temperature b. Stored in frozen state (1) Frequently used reagents should be frozen in aliquot (2) Avoid repeatedly freezing-thawing (3) Certain enzymes are very sensitive to freezing L-SP c. Frozen in glycerol Protein stored in -20℃ in 50% glycerol d. Avoid light and microbe How to store your reagents or enzymes Mixed well Juang RH (2005) EPA ■ 細胞內外酵素分佈 Cellular distribution of enzymes Protein in expressing Enzymes in the cell Organelle Cytosolic enzymes Endogenous organelle enzymes Imported enzymes Secreted enzymes Continuous secreting Regulatory secreting Soluble enzymes Membrane proteins Juang RH (2005) EPA ■ 各種酵素的安定性都不同 Enzyme stability ● Reasons enzyme lost its activity: ● Protein Denatured ● Active site destroyed ● Protease proteolysis ● Enzyme Inhibitor ■ Physical denaturation Physical/chemical denaturation Chemical reaction Inactivated by degradation Natural or synthetic inhibitors Heating, freezing, foaming, adsorbing ■ Chemical denaturation Extreme pH, oxidation, heavy metals Juang RH (2005) EPA ■ 蛋白脢的專一性及其抑制劑 Protease families Family Example CarboxyMetal Protease peptidase A Serine Chymotrypsin Trypsin Protease Mechanism Zn 2+ E72 H69 H196 S195-OH57 D102 C25-S- Cysteine Protease Papain H195 Aspartyl Protease Pepsin Renin D215 H2O D32 Specificity Inhibitor Nonpolar EDTA EGTA Aromatic Basic DFP TLCK TPCK Nonspecific PCMB Leupeptin Nonspecific Pepstatin Juang RH (2005) EPA
Similar documents
3.3 離子交換法Ion exchange 3.3.3 緩衝液與層析系統Buffer system
■ 質子可以吸著或脫離一基團
Proton： the smallest and most abundant particle in the living cell
controlling the pH and the charge property of a molecule
lone pair
electrons