【Purpose】

1. Through learning the theories and techniques of biochemistry and correlative subjects, be familiar with and further grasp experimental skills on the extraction, separation, purification and assay of certain enzyme and master its physical and chemical properties.
2. Form the ability to design the experiment on the whole perspective level, and further improve the ability to analyze and solve problem.
3. Cultivate co-operative spirit and economical while high efficiency habit in the scientific research.

【Requirement】

1. Use bread yeast as the original material to extract and condense carboxypeptidase Y, assay its biochemical properties.
2. Through gathering relevant published papers, analyze and design the experiment and write down the feasibility report.
3. The report includes the purpose, the meaning, and the recent researches on this experiment abroad and in china, methods and also technical means, while the analysis of possibility and the expected results of this experiment should be added.
4. One month before the beginning of this experiment, the draft of experiment report should be delivered to the guidance teacher so that the teacher could look through and prepare for it.
5. The draft of the report should be based on at least 10 published papers, among which are at least 2 papers published in recent 5 years, including new technology or new method in the research of application on biomacromolecules.
6. The quotation of published papers should follow the form in which includes the author, article name, journal name, volume, page and reference books.
7. In this experiment, every group consists of four students. In part of the experiment, several groups need to cooperate
8. Experiment report should include principle, reagent, apparatus, equipment, protein product ratio, activity ratio and final product ratio in each step of extraction, separation and purification; Following the content of experiment, provide the result and related figure or chart and discuss the experiment on the whole perspective.

【Contents】

1. Assay of carboxypeptidase Y activity
⑴ Method of enzyme activity assay.
⑵ The definition of enzyme activity unit
⑶ The definition of specific activity
⑷ Method of lowry method to quantitate protein
2. Extraction of carboxypeptidase Y in baker yeast.
⑴ Give every group 1.5kg of fresh bread yeast
⑵ Extract by some methods such as solubility
⑶ Activation of carboxypeptidase Y
3. Separation and purification of carboxypeptidase Y
⑴ Separation carboxypeptidase Y from above-mentioned solution by various methods.
⑵ Assay of protein purification before purification.
⑶ Assay of molecular weight
⑷ Assay of isoelectric point
4. The physical and chemical properties of carboxypeptidase Y
⑴ Validate purification of carboxypeptidase Y by two methods.
⑵ Assay the molecular weight of carboxygpeptidase Y
⑶ Assay the isoelectric point of carboxygpeptidase Y
5. The enzymatic properties of carboxypeptidase Y.
⑴ Calculate Vmax and Km of carboxypeptidase Y
  ⑵ First make sure the optimum pH of carboxypeptidase Y.
  ⑶ First make sure the optimum temperature
  ⑷ Propose at least two inhibitors of carboxypeptidase and prove the inhibition style
⑸ Propose the activators of carboxypeptidase Y and then prove them
⑹ Prove the stability of carboxypeptidase Y

【Principle】

Carboxypeptidase Y is a kind of proteases in bread yeast. It has the ability to remove most amino acid residue, including that of proline, from the C-termini of proteins and peptides. These unique properties render a useful tool in C-termini analysis. The enzyme is quite different from the pancreatic carboxypeptidases A and B in many aspects. Carboxypeptidase Y is an acidic glycoprotein without metals.
Since carboxypeptidase Y was found in 1967, its physical and chemical properties, structure, application had been researched. The enzyme is a glycoprotein having about 16 residues of glucosamine and about 15%hexose. The enzyme is rich in acidic amino acid, so the isoelectric point is 3.6. Carboxypeptidase Y is an acidic protein with a molecular weight of approximately 61000. The absorptivity of the enzyme that is calculated using the A _280nm of a 1%solution is 15. Carboxypeptidase Y is a diisopropyl-phospho-fluoridate (DFP)-sensitive enzyme. Its inactivation by DFP is accompanied by the formation of 1 mole of labeled serine per mole of enzyme. Carboxypeptidase Y is also sensitive to metal ions such as Cu ²⁺ ,Hg ²⁺ ,Fe ³⁺ ,Fe ²⁺ ,Mg ²⁺ .etc. Therefore, one must take the general precaution of preventing it from contacting with metal ions in purifying and handling carboxypeptidase Y. The enzyme is relatively stable in the presence of protein denaturants or certain solvents. About 80%of the activity is retained after incubation of the enzyme with 6M urea at 25℃ for 1 hr. In the presence of 10% methanol, the enzyme is completely stable at pH 5.5～8.0 for 8 hr at 25℃. Aqueous solu­tion of the enzyme is stable for 8 hr in the pH range 5.5～8.0 at 25℃ and for 2 hr in the pH range 6～8 at 37℃. At higher temperatures, the enzyme is most stable at pH 7.0. Its activity is quickly lost below pH 3 or above 60℃. The enzyme in saturated (NH ₄ ) ₂ SO ₄ , can be stored at -20℃ for a long time. 1% aqueous solu­tion of the enzyme is stored at - 20℃. The frozen solutions show no loss of activity for at least 2 years. The activity of greatly diluted solutions (below 0.1 mg/ml) is quickly lost, Re­peated freezing and thawing of solutions of the enzyme, or prolonged storage at room temperature, can lead to autodigestion.
Carboxypeptidase Y is stored as zymogen in yeast. It can be separated from other proteins in yeast by the above-described properties. First, the yeast cell autolyze with organic solvents, and activate the enzyme under pH5.0 in order to convert zymogen into active enzyme. Secondly, chromatography on DEAE-cellulose is used to remove other proteins. Finally, chromatography on DETA-Sephadex A-50 and Sephadex G-150 are applied to purify further the enzyme, and the enzyme is identified through polyacrylamide gel electrophoresis.
Carboxypeptidase Y possesses the activity of esterase and peptase that can be assayed using N-acetyl-L-tyrosine ethyl ester (ATEE) and benzyloxycarbonyl-L-phenylalanyl-L-leucine (CBZ-Phe-Leu) as substrates respectively. Spectrophotometric method is applied to assay in general.

【Materials】

1. Apparatus
Vacuum pump of recycle water; Ultraviolet detector, recording machine; UV-Visible Spectrophotometer; Stepwise mixer (500ml); 721- Spectrophotometer; pH meter; High speed centrifuge; Refrigerator; Constant temperature water boiler; G-3 Core sand funnel (500mL); Fraction collector; Constant speed pump; Disk electrophoresis apparatus; Chromatography column (2.6×50cm) (1.0×100cm) (0.6×30cm); Dialytic bag.
2. Reagents
⑴ DEAE-ion exchange cellulose (DE-32): Weigh out the appropriate amount of DE-32 for the required bed volume of your column. Swell it in distilled water completely, and then decant the small particles that can not precipitate, then suction the basic liquid, wash it to pH7.0 with distilled water; Soak the resin with 0.5mol/L hydrogen chloride for 30mins, and then suction the solution and wash it to pH7.0 with distilled water; Soak the resin with 0.5mol/L sodium hydroxide solution for 30～60 minutes, and then suction the solution and wash it to pH7.0 with distilled water. Equilibrate with phosphate buffer required before stuffing column. The used DE-32 should be soaked in 0.5mol/L sodium hydroxide-0.5mol/L sodium chloride solution and washed completely with distilled water before using the method as above described.
⑵ DEAE-Sephadex A-50: The process of disposition is the same as DE-32 except that 0.1mol/L of hydrogen chloride and sodium hydroxide solution are used to soak gel for 20mins respectively.  
⑶ Sephadex G-150: The method is the same as experiment 5
⑷Tetramethylethylenediamine (TEMED); Tris (hydroxymethyl)aminomethane; sodium dodecyl sulphate ( SDS ); Acrylamide；Bisacrylamine; Coomassie Brilliant Blue G250
⑸ Ampholyte (pH3～10) ; Fetal bovine serum (FBS)
⑹ 0.5mmol/L Benzyloxycarbonyl-L-phenylalanyl-L-leucine (CBZ-Phe-Leu) : Weigh 5.17mg of CBZ-Phe-Leu，dissolve it into 0.05mol/L pH6.5 phosphate buffer solution and set the volume at 25ml.
  ⑺ 1mmol/L N-acetyl-L-tyrosine ethyl ester(ATEE): Weigh 6.25 mg of CBZ-Phe-Leu, dissolve it into 0.05mol/L pH7.5 phosphate buffer solution and set the volume at 25ml.
  ⑻ Nessler's reagent: Dissolve 10g hydrargyri iodidum rubrum and 7g kalium iodidum in 10ml distilled water, add 24.4g potassium hydroxide to a volumetric flask containing 70ml distilled water. After cooling, add slowly solution with hydrargyri iodidum rubrum and kalium iodidum to volumetric flask under shaking constantly. Set the volume at 100ml and mix completely. Use it after at least two days. Store the solution into brown flask in darkroom.
⑼ NaOH; HCl; Chloroform; Toluene; Fresh bread yeast (1.5kg); Solid (NH ₄ ) ₂ SO ₄ ; Na ₂ HPO ₄ ·12H ₂ O; NaH ₂ PO ₄ ·2H ₂ O; Ethanol; Distilled water; Methanol; Trichloroacetic acid; Acetone      

【Procedures】

1. Purification procedure
⑴ Autolysis: 1.5kg of fresh bakers’ yeast is crumbled and mixed with 330 ml of chloroform. The mixture is occasionally kneaded with a spatula or glass rod, until the yeast has liquefied (30～60 min). When the cooled yeast has been delivered, the container of the mixture is immersed in a water bath at 30℃ during kneading of the mixture. After liquefaction, add 600ml of distilled water, the mixture is stirred. The pH of the mixture is adjusted to 7.0 with 1 M NaOH under vigorous stirring, and the whole is kept at 25℃ for approximately 2 h. The pH is readjusted to 7.0 with 1 M NaOH and the mixture is left at 25℃ for an additional 16 hr to completely autolyze.
Removal of cell debris：The autolytic mixture is centrifugated at 4300 g for 15 minutes and the residue is discarded. The supernatant solutions are combined (about 1.4 liters).
⑵ Fractionation by ammonium sulfate：Solid (NH ₄ ) ₂ SO ₄ (313 g/L，25℃，0.5 saturation) is added gradually with stirring to the combined supernatant solutions, while the pH is maintained at 7.0 with 1M NaOH. Supernatant solution is put for 2h, and then the precipitate is removed by centrifugation at 8400g for 20 minutes. Solid (NH ₄ ) ₂ SO ₄ (302 g/L of the initial solution，25℃，0.9 saturation) is added to the combined supernatant solutions, while the pH is maintained as before. After the (NH ₄ ) ₂ SO ₄ is completely dissolved, the suspension is left overnight, then the precipitates are collected by centrifugation at 13,000 g for 30 minutes.
⑶ Activation: The precipitate is dissolved in 300ml of 0.05 M sodium acetate buffer, pH 5.0, and adjusted to pH 5.0 with 1 M acetic acid under stirring, then incubated at 25℃ for an additional 18～20h with a few drops of toluene added.
⑷ Chromatography on DEAE-cellulose: The activated enzyme solution (600ml) is dialyzed against 0.01 M sodium phosphate buffer, pH7.0. Afterwards, add solid NaCl to make the solution 0.1M in NaCl and continue to dialyze until NH ₄ ⁺ is cleared away which is check using Nessler's reagent. The solution is then poured onto a column of DEAE-cellulose (2.6×50cm) equilibrated with 0.01 M sodium phosphate buffer, pH 7.0, containing 0.1 M NaCl. After all the solution passed through, elution is performed with a linear increase in the NaCl concentration from 0.1 M to 0.42 M (each chamber, 280 ml) at a flow rate of 30 ml/h，4mL/tube. Collect the elution using fraction collector and determine enzyme activity.
⑸ Chromatography on DETA-Sephadex A-50: The enzyme fraction is collected and is precipitated by dialysis against the batch of saturated (NH ₄ ) ₂ SO ₄ , and then it is adjusted to pH 7.0 at 5℃ over 10h. The precipitate is col­lected by centrifugation at 20000 g for 20 minutes. The precipitated is dissolved in a minimum volume of 0.01 M sodium phos­phate, pH 7.0, containing 0.15M NaCl, and then dialyzed against this buffer with frequent changes. The enzyme solution is applied to a column of DEAE-Sephadex A-50 (0.6×30 cm) equilibrated with the same buffer. After the column has been washed with the starting buffer, elution is performed with a linear increase in the NaCl concentration from 0.15 M to 0.5 M (each chamber, 120 ml) at a flow rate of 20 ml/h，3ml/tube. Collect the elution using fraction collector and determine enzyme activity.
⑹ Chromatography on Sephadex G-150: Pooled fractions are dialyzed against saturated (NH ₄ ) ₂ SO ₄ , as in step （5）, The enzyme precipitated is collected by centrifugation at 20,000 g for 30 min. The precipitate is dis­solved and dialyzed as in step（5）. The enzyme solution is applied to a column of Sephadex G-150 (1.0×100cm) equilibrated with 0.01 M sodium phosphate, pH 7.0, containing 0.15 M NaCl. The enzyme is eluted as in step（5）at a flow rate of 4.5 ml/h，1ml/tube. Carboxypeptidase Y gives a single peak, the pooled fraction is concentrated with (NH ₄ ) ₂ SO ₄ as described above and stored at refrigerator.
2. Assay of enzyme activity
⑴ Assay of esterase activity: This method is based on titrimetric measurement of the release of protons or upon spectrophotometric measurement of the change in ultraviolet absorbance that occurs as a result of the enzymic hydrolysis of a,N-acetyl-L-tyrosine ethyl ester (ATEE). Spectrophotometric method is as follows:
Two 1-cm cuvettes, one containing 2ml of substrate solution (1mM ATEE dissolved in 0.05 M sodium phosphate, pH 7.5) and the other containing 2 ml of buffer (0.05 M sodium phosphate, pH 7.5) are placed in a double-beam spectrophotometer and allowed to equilibrate at 25℃ for 5 min. The wavelength used is 237 nm for ATEE. The reaction is initiated by adding 10µl of the enzyme solu­tion to the reaction cuvette, and the change in absorbance is recorded at 30 sec intervals. If △A _237nm >0.400, the enzyme should be diluted. Activity unit and specific activity are calculated using equations as follows.
One unit of activity is defined as the amount of enzyme that makes 1µmol of ATEE hydrolyze, and the hydrolysis of 1µmol of ATEE causes decreases in absorbance of 0.1 per minute.

 

△A _237nm is the change value of absorbance at selected time intervals; ε is the amount of added enzyme (µg); 1000 is transition value from microgramme to milligramme.
⑵ Assay of peptase activity: This method is based on the rate of the enzymic hydrolysis of benzyloxycarbonyl-L-phenylalanyl-L-leucine (CBZ-Phe-Leu ). The rate of the reaction can be measured either with the colormetric ninhydrin or spectrophotometric method by the decrease in absorbance at 224 nm. Operation is the same as assay of esterase activity. Substrate solution is 0.5mM CBZ-Phe-Leu dissolved in 0.05 M sodium phosphate, pH 6.5). One unit of activity is defined as the amount of enzyme that makes 1µmol of CBZ-Phe-Leu hydrolyze, and the hydrolysis of 1µmol of CBZ-Phe-Leu causes decreases in absorbance of 0.2 per minute.
3. Determination of protein content
Folin-phenol method. (the same as experiment 2.)
4 . Test of purify
Polyacrylamide gel electrophoresis ( PAGE) method (the same as experiment 6)
5. Study on physical, chemical and enzymatic properties
According to the results of purification and activity assay, the procedures designed by students themselves should be applied to research correlative properties of this enzyme.
【 Results 】

Procedures	Volume (ml)	Total protein (mg)	Activity unit	Specific activity (unit/mg)	Recovery ratio (%)
1. Autolysis			----	-----	-----
2. Fractionation by ammonium sulfate			-----	-----	-----
3. Activation					100
4. Chromatography on DEAE-cellulose
5. Chromatography on DETA-Sephadex A-50
6. Chromatography on Sephadex G-150

【Questions】
1. Conclude and expound the knowledge of biochemistry involved in the experiment.
2. Propose some feasible measures to optimize the experiment according to your experience from the experiment.