兔磷酸激酶实验报告英文.docx

1、兔磷酸激酶实验报告英文Isolation and Enzymatic Analysis of Creatine Kinase of Rabbit MuscleI. IntroductionI. Creatine kinaseCreatine kinase usually exists in cytoplasm and mitochondria of organs like animal heart, muscle, brain and so on. It is an important kinase which relates to the energy operation of cells,

2、 muscle contraction and ATP regeneration. Creatine kinase reversibly catalyze phosphoryl transfer reactions between creatine and ATP.There are four forms of creatine kinase isoenzyme: MM, BB, MB, MiMi. MM mainly exists in all kinds of muscle cells, BB mainly exists in brain cells, MB mainly exists i

3、n myocardial cells while MiMi exists in the mitochondrial inner membrane. MM, BB, MiMi are plasma creatine kinase.Phosphorylation oxidative occur in the mitochondrial of cell, which produce ATP. But the high-energy material which animals use to store energy is creatine phosphate. Energy storage and

4、transportation is complete via creatine phosphate shuttle, which include MM and MiMi. ATP produced by oxidative phosphorylation can be directly sent to the locatioin of MiMi in the outer mitochondrial membrane. MiMi transfer the high-energy phosphate bond in ATP to creatine into ADP and creatine pho

5、sphate energy material. Creatine phosphate spread in cell and reach myofibrils. The M-zone of myofibrils combines MM, thus, when muscle contracts, large amounts of ATP are required for energy supply and MM catalyst creatine phosphate into ATP. The creatine produced by the reaction will get to mitoch

6、ondria and get re-phosphorylated.Creatine kinase has important physiological functions and medical applications.Muscle-type creatine kinase molecule is composed of two identical subunits dimer. In catalytic process the smallest functional unit of the enzyme is subunit which have catalysis function i

7、ndependently. According to the creatine kinase primary structure measured in rabbits, human, chicken, mouse recently, M-type subunit consist of 387 amino acid residues and has the molecular weight around 43 000ug. There are 8 sulfhydryl but has no disulfide bond. Natural creatine kinase molecule is

8、a compact globular structure. According to the study of optical rotatory dispersion, the subunit of this enzyme has about 25% -30% of the -helix, 15% of the -sheet.2. Purification of creatine kinaseThere are many ways of purifying creatine kinase,and the purification method used in this study is imp

9、roved Kuby method.3. Measurement of creatine kinase activitiesThere are many ways of measuring the activities of creatine kinase, and we use pH-colorimetry in this study.pH-colorimetryWhen creatine kinase is catalyze forward reaction, with the transfer of Phosphoryl from ATP to creatine, the equimol

10、ar H+ is produced, whose optimum pH is 7.59.0. Within this context, the generation rate of H + measured can be used as indicators of activity. In this study, thymol blue is used as a pH indicator. The activities of creatine kinase is measured by pH-colorimetry on spectrophotometer, which is monitori

11、ng changes in absorbance under the wavelength of 597nm. In the cuvette, H + produced by the enzyme-catalyzed reaction lower the pH value of solution. The color of substrate solution gradually change from deep purple red yellow green and lower the Absorbance value of A597. In the pH-colorimetry, the

12、substrate solution requires to be new.4. Measurement of creatine kinase solution 280nm light absorptionMethod for protein measurementSensitivityTimePrincipleInterferenceCommentsSpectrophotometric(A280)Moderate50-1000 gRapid5-10 minAbsorption of 280nm light by aromatic residuesPurines, pyrimidines, n

13、ucleic acidsUseful for monitoring column eluents. Nucleic acid absorption can be corrected. Nondestructive to protein samples. Varies with proteins.II. Experimental1. Reagents and Equipments1) Reagents1. Preparation of crude extract:0.01M KCl, NH4Cl, 5M NH4OH, anhydrousethanol, pH8.5-9.0 2M MgSO4a.

14、KCl：0.01mol/L,200ml. (0.15g KCl add water to 200ml)b. NH4OH：1.7mmol/L,1000ml.(take 0.12ml NH3H2O,add H2O to 1000ml) c. NH4Cl d. Anhydrous ethanole. MgSO4：2.0mol/L，pH8.5，20mlf. MgAC2：0.07mol/L，pH9.0，100mlg. Tris-HCl：0.1mol/L，pH8.0，1000ml（Tris 12.1g, HCl 23ml, pH为8.0，add water to 1000ml）h. Salt2. Chro

15、matographya. 48mmol/L creatine kinase solutionb. 0.1mol/L MgAC2c. 0.1 Thymol blue (100mg Thymol blue，dissolve with 20ml ethanol and 60ml ddwater).d. 0.1mol/L pH9.0 Gly-NaOH buffere. ATPf. 0.1mol/L NaOH g. 0.07M. MgAC2 pH9.0h. 0.01M Tris-HCl pH8.02) Equipments1. Preparation of crude extract:Knife, me

16、at chopper, plastic basin, 1/100 balance, High-Speed diffuser, refrigerator;100ml beaker, 25ml beaker, 100ml measuring cylinder,250ml measuring cylinder;Pippet, burette, glass stick;0.5ml Ep tube, 1.5ml Ep tube;centrifugal machine, 50ml centrifugal tube, stainless steel scoop, mortar;drinking paper,

17、 accurate pH test paper2. Measurement of enzymes activities:SPECORD200 Spectrophotometer, Pippete, 50ml beaker, burette.3. Measurement of protein solutions:SPECORD200 Spectrophotometer.2. Procedure1) Preparation of crude extract1. With a sharp knife, cut the muscle into cubes 4 to 5 cm wide from the

18、 ice-cold rabbit muscle.2. Quickly weigh about 50 g of the muscle cubes and suspend the cubes in 140 mL of 0.01 mol/L KCl at 0 C.3. Homogenize 140 mL of the suspension in a glass beaker with a blender (turn on the blender at high speed for 20 seconds) and stir them with a glass stirring rod at 0 C f

19、or 15 minutes.4. Centrifuge the homogenate for 10 minutes at 10000 r/min at 0 C. 5. Carefully decant the supernatant and measure the volume of the supernatant (V1) (keep 0.6mL for protein assay at 20 C). Discard the pellet.6. Add ground ammonium chloride (NH4Cl) to make 0.1 mol/L solution and then a

20、djust pH to 9.0 with 5 mol/L ammonium hydroxide (NH4OH), keep stirring the solution at 0 C for 30 minutes.7. Add 1-fold V1 of cold 95% ethanol and stir the solution at 20 C for 30 minutes.8. Centrifuge the solution for 10 minutes at 10000 r/min at -8 C. Discard the pellet and measure the volume of t

21、he supernatant (V2) (keep 0.6 mL for protein assay at 20 C).9. Take the supernatant (V2) and add VA (mL) of 2 mol/L MgSO4 (pH 8.5) to a final concentration of 0.03 mol/L.10. Add 1-fold VA of cold 95% ethanol and stir the solution at 0 C for 30 minutes.11. Centrifuge the solution for 10 minutes at 10

22、000 r/min at -8 C. Pour off and discard the supernatant. 12. Suspend the pellet with 1/10 V1 0.07 mol/L MgAC2 (pH 9.0). 13. Stir the suspension at 0 C for 30 minutes. Centrifuge the suspension for 10 minutes at 12000 r/min at 0 C. Pour off and discard the pellet. 14. Measure the volume of the supern

23、atant (V3) (keep 0.6 mL for protein assay at 20 C).15. Adjust the pH of the supernatant to 8.0 with 1 mol/L NaOH and add VB of cold 95% ethanol to a final concentration of 36% in an ice-salt bath. Calculate the VB using: (V3-the volume of MgAC2)x0.5+ VB/(V3+ VB)= 0.36.16. Stir the solution at 0 C fo

24、r 30 minutes. Centrifuge for 10 minutes at 12000 r/min at -8 C. Pour off and discard the pellet. Measure the volume of the supernatant (V4) (keep 0.6 mL for protein assay at 20 C).17. Add VC cold 95% ethanol to a final concentration of 50% in an ice-salt bath. Calculate the VC using: (V4x0.36 + VC )

25、/ (V4 + VC )= 0.6.18. Stir the solution at 0 C for 30 minutes. Centrifuge for 10 minutes at 12000 r/min at -8 C. Pour off and discard the supernatant. Suspend the pellet in about 5 mL of 0.01 mol/L Tris-HCl (pH 7.5). Centrifuge for 10 minutes at 12000 r/min at 0 C. Measure the volume of the supernat

26、ant (V5) (keep 3-4 mL for protein assay at 20 C). 19. Store the V5 at 20 C overnight.2) Chromatography1. Q Sepharose High Performance column has been prepared for your use. Before starting a run, the column has to be equilibrated. This is done by pumping 5 column volumes of start buffer (buffer A: 0

27、.01 mol/L Tris-HCl , pH 7.5) through the column. 2. Measure out 45 mL of V5 for application to the column, then wash the column with 2 column volumes of buffer A and elute the sample with buffer B (0.01 mol/L Tris-HCl, 0.3 mol/L NaCl , pH 7.5) in a linear gradient, flow rate of the column is about 1

28、 mL/min.3. Collect 1 mL fractions in test tubes in a fraction collector. Transfer the contents of every other fraction to a quartz cuvette and measure the absorbance at 280nm or use a UV monitor. Record the A280 values in your notebook. When you begin to detect protein eluting from the column (A280

29、0), measure the A280 of every fraction.3) Measurement of Enzyme Activities1. The detection of the specific activity of the creatine kinase is based on the pH-colorimetry. It is rather sensitive for the advanced spectroscopy to monitor the alteration of the color of Thymol blue. 2. The way to calcula

30、te the specific activity is according to the following equation:1.3 is a converting coefficient. U stands for specific activity. VA stands for the volume of substrate (VA=1.0mL). VB stands for the volume of enzyme solution (VB=0.01mL). C is the concentration of enzyme solution, and it is determined

31、by the absorbance of the solution at 280nm.3. Fill the specific activity of the creatine kinase in each fraction in the form bellow.Fraction Protein Concentration(mg/mL) Specific Activity Fold Purification Recovery (%) 4) SDS-PAGEThe samples obtained in each step of crude extraction and the final pr

32、oducts from ion-exchange chromatography are loaded onto sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). The stacking gel was made of 3% acrylamide, and the separating gel was made of 12.5% acrylamide. III. Experimental record1. The volume in each purification stageChart 1. Volume of the protein.V1V2V3V4V546.8 ml83.1 ml4.98 ml5