Enzymology - Kau
Kingdom of Saudi ArabiaKing Abdulaziz UniversityGirls Collage of ScienceBiochemistry DepartmentEnzymologyPractical ManualBIOC231Name:Computer No.:Section:1
ContentsLab #ExperimentPage1Effect of Amylase activity on Starch32Determination of α-amylase activity83Effect of pH on amylase activity124Investigation effect of temperature on the activity of lipase155Hydrolysis of sucrose by yeast β-Fructofuranosidase196Determination of Hydrolyzed Sucrose Solution bybenedict quantitative method237Estimation of lipase activity278Indirect estimation of lactate dehydrogenase309Detection of Enzymes3310Detection of Enzyme mixture362
Experiment 1:Effect of Amylase activity on StarchDefinition of enzymes: enzymes are biological catalysts. They greatlyenhance the rate of specific chemical reactions that would occurvery slowly.Starch which is the storage form of glucose in plant. Starch consist of1- Amylose1-4 α- glycosidic linkage2-Amylopectin1-6 α- glycosidic linkage3
Contents of Saliva:In animals, saliva is produced in and secreted from the salivaryglands. It is a fluid containing: m, 1.2-2.8 mmol/L calcium, 0.08-0.5 mmol/Lmagnesium, 5-40 mmol/L cloride, 2-13 mmol/L bicarbonate,1.4-39 mmol/L phosphate) ides and glycoproteins; Antibacterial compounds (thiocyanate, hydrogen peroxide,and secretory immunoglobulin A) various enzymes. The major enzymes found in human salivaare alpha-amylase, lysozyme, and lingual lipase. Amylasestarts the digestion of starch before the food is evenswallowed. It has pH optima of 6.7-7.4. Human aryA B,N-NAD(P)Hlactoperoxidase,superoxide dismutase, glutathione transferase, glucose-6phosphate isomerase, and tissue protein. The presence ofthese things causes saliva to sometimes have a foul odor.Healthy people produce about 1.5 L of saliva per day.Amylase:found in two forms:1. α-amylase (in saliva and pancreatic juice) which isendoglycosidase that attack starch randomly. Inactivated by theacidity of the stomach.2. β-amylase (from plant origin) which is exoglycosidase cleavesmaltose from the non-reducing end to produce β-maltose4
Principle:When we want to measure enzyme activity either we measure thedecrease in the substrate concentration or the increase in the sepH 6.4-7.2 Cl reducing sugarIndicator I2Indicator FehlingBlue colorRed copper oxide pptOther uses of amylase in industry:It is used in clarification of fruit juices. The turbidity present in naturalbeverages is due primly to the presence of starch and cellulosemolecules too large to be completely soluble. Amylase hydrolysis thesemolecules to glucose which are more water soluble.Reagents: Starch 1% solution in 0.3% aqueous sodium chloride Freshly prepared; iodinated potassium iodide solution. Amylase5
Procedure:Prepare 2 test tubes which contain the following:Test tubeABAmylase-1 mlStarch1 ml1 mlAllow the tubes to stand for 30 min in water bath (37 C - 40 C)Iodine solution1-2 drops1-2 dropsReferences:1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 19782- Harvey,R and Champe,P. Lippincott biochemistry, london.2005.6
Results SheetExperimentObservationCommentA( Starch only)B(Starch Amylase)7
Experiment 2: Determination of α-amylase activityHistory:α-Amylases (EC 3.2.1.1) is an enzyme of glycoside hydrolases mainlyproduced in the salivary glands and pancreas, play a well-known role inhydrolyzing a-1,4- glucosidic bonds between glucose in starch ( consistsof two types of polysaccharide amylose, amylopectin) and maltose isrelease. Elevated level of α-Amylases in serum can be used as markersfor clinical diagnosis of diseases, e.g. Pancreatitis. When the pancreas isdiseased or inflamed, amylase releases into the blood.Principle:The α -amylase activity is measured using a colorimetric method with3,5-dinitrosalicylic acid (DNS) reagent. In this method, starch by α –amylase is converted into maltose. Maltose released from starch ismeasured by the reduction of 3,5-dinitrosalicylic acid.Starch H2O α-AmylaseMaltose (reducing agent)8
Maltose reduces the pale yellow coloured alkaline 3, 5-Dinitro salicylicacid (DNS) to the orange- red colored. The intensity of the color isproportional to the concentration of maltose present in the sample.This intensity change in color is measured using a spectrophotometer asthe absorbance at 540nm wavelength. Wave length is set to 540 nmbecause it is the region where orange-red color absorbs.This procedure applies to all products that have a specification for αamylaseReagents: 0.02 M Sodium phosphate buffer 1% Starch 2 N Sodium hydroxide Sodium potassium tartrate tetrahydrate9
Dinitrosalicylic acid color reagent Standard Maltose Stock Solution Amylase enzymeProcedure:Adjust spectrophotometer at 540 nm and 25 C.TubeStarchEnzymeDistell waterMaltoseTest0.5 ml0.5 ml-Blank0.5 ml0.5 ml-Standard0.5 ml0.5 mlMix well and incubate at 25 C for 3 minutesDinitrosalicylicacid colorreagent1 ml1 ml1 mlIncubate all tubes in a boiling water bath for 5 minutes. Cool to roomtemperature10mlDistell water10ml10mlMix well and read the increase in optical density at 540 nm againstblankDetermine micromoles maltose released from standard or standard curveCalculationEnzyme activity OD (test) x concentration of St (µmoles) x dilution of enzymeOD (st) x incubation time (3 min)Enzyme Unit µmoles maltose formed / min/0.5 ml (x 2) µmoles maltose formed / min/ml10
Results Sheet11
Experiment 3:Effect of pH on amylase activity- The effect of pH on α-amylase activity will be studied- Enzymes are affected by changes in pH. The optimum pH valueis defined as the pH at which the enzyme rate of reaction (enzymeactivity) reach the maximum activity (V max)- Deviation in pH from the optimum cause decrease in enzymecatalytic activity- Extremely high or low pH values generally result in complete lossof activity for most enzymes.- The enzyme stability is depending on the optimum pH. Eachenzyme has a region of optimum pH for stability.- The optimum pH can be determined by incubating the enzyme indifferent incubation media containingdifferent pHbufferrange from 1.5 –10. The enzyme activity will be calculated at eachpH at which the enzyme will be incubated. Plot a curve of enzymerate of reaction(enzyme activity) against the different pH atwhich the enzyme catalytic reactions are incubated.- From the curve, the optimum pH which givethe maximumactivity of the enzyme will be determined12
Procedure:TubeStarch (PH 1.5)Starch (PH 6.9)Starch (pH 10)EnzymeDistill waterMaltose10.5 ml0.5 ml-20.5 ml0.5 ml-30.5 ml0.5 ml-Blank0.5 ml0.5 ml-Standard0.5 ml0.5 mlMix well and incubate at 25 C for 3 minutesDinitrosalicylicacid color reagent1 ml1 ml1 ml1 ml1 mlIncubate all tubes in a boiling water bath for 5 minutes. Cool to roomtemperatureDistill water8 ml8 ml8 ml8 ml8 mlMix well and read the increase in optical density at 540 nm againstblankDetermine micromoles maltose released from standard or standard curveCalculationEnzyme activity OD (test ) x concentration of St (µmoles) x dilution of enzymeOD (st) x incubation time (3 min)Enzyme Unit µmoles maltose formed / min/ 0.5 ml enzyme (x 2) µmoles maltose formed / min/ ml enzyme13
Results Sheet14
Experiment 4:Investigation effect of temperature on theactivity of lipaseThis practical gives you a chance to: investigate how lipase activity changes with temperature consider how indicators can help us to follow chemical reactions.Procedure1- Label a test tube with the temperature (25 C- 40 C70 C).2- Add 5 drops of phenolphthalein to the test tube.3- Measure out 5 ml of milk using a measuring cylinder (orsyringe) and add this to the test tube.4- Measure out 7 ml of sodium carbonate solution usinganother measuring cylinder (or syringe) and add this tothe test tube. The solution should now be pink.5- Place a thermometer in the test tube. Take care as theequipment could topple over.6- Place the test tube in a water bath and leave until thecontents reach the same temperature as the water bath.7- Remove the thermometer from test tube and replace itwith a glass rod.8- Use the 2 ml syringe to measure out 1 ml of lipase fromthe beaker in the water bath for the temperature you areinvestigating.9- Add the lipase to the test tube and start the stop clock/stopwatch.10- Stir the contents of the test tube until the solution losesits pink color.11- Stop the clock/ watch and note the time in a suitabletable of g15
Results SheetExperimentObservationCommentA (at 25 C)B (at 40 C)C (at 70 C)16
QUESTIONS1- When fat breaks down, what is produced?2- Use this information to explain why the phenolphthalein changescolour?3- What is the effect of temperature on the time taken for lipase tobreak down the fat in milk?4- Why does the temperature affect the action of lipase in this way?5- What is the difference between a ‘time taken’ and a ‘rate ofreaction’ curve for this investigation?17
ANSWERS18
Experiment 5: The hydrolysis of sucrose by yeast e is a glycosidase found in yeast. It catalyses thehydrolysis of sucrose to glucose and fructose. The enzyme is also knownas invertase or sucrase, but these names are no longer used.The substrate sucrose is a non-reducing sugar, whereas the productsformed are both reducing sugar. Therefore the reaction can be followedby the estimation of the quantity of reducing sugar formed. Between theseveral methods which can be used for such estimation, Benedictquantitative method was utilized.Benedict quantitative reagent is composed of:1- Copper sulphate: to provide the oxidizing Cu 2ions.2- Sodium carbonate: to provide the alkaline medium necessary forthe formation of the highly reactive reducing sugar 1-2 endediol.3- Sodium citrate: combines with Cu carbonate to prevent itsprecipitation by forming a slightly soluble complex with cupricions (Cu 2ions). This complex dissociates slowly to give asufficient supply of Cu 2 ions.4- Potassium thiocyanate (KSCN): reacts with cupric ions to giveCu(SCN)25- Potassium ferrocyanide (K4Fe(CN)6): prevents the re-oxidation ofthe formed cuprous thiocyanate (CuSCN) to cupric thiocyanate.19
The reaction takes place as fallow:1- The enolization of reducing sugar in alkaline medium to give ahighly reactive reducing compound, which is 1-2 endediol2- Formation of cupric carbonateNa2CO3 CuSO4 CuCO3 Na2SO43- Formation of sodium cupric citrate complex:20
4- Ionization of sodium cupric complex5- Reaction of KSCN with Cu 2ions:Cu 2 KSCNCu(SCN)2Cupric thiocynate (blue)6- Reduction of Cu(SCN)2 by 1,2 enediol to cuprous thiocynate:boilCu(SCN)2CuSCN21
Procedure:A- Hydrolysis of sucrose by yeast β-FructofuranosidasePrepare five tubes containing the following mixtures:Tube12345Sucrose 0.3M10 ml8 ml6 ml4 ml2 mlD.W02 ml4 ml6 ml8 mlBuffer pH 4.56 ml6 ml6 ml6 ml6 ml4 ml4 mlPre-incubate at 37C for 5 minYeastsuspension4 ml4 ml4 mlIncubate for 15 min1% NaOHFinal conc. Ofsucrose2 ml2 ml2 ml2 ml2 ml150120906030Note:The yeast must be added to each tube at a constant time intervals, i.e. tube1 at time 0, tube 2 at 2min etc. This will enable the incubation time to bemeasured exactly and ensures that each tube is incubated for the sametime. Incubate each tube for exactly 15 min. Stop the reaction by theaddition of 2ml of 1% sodium hydroxide. This will be at 15 min for tube1, 17 min for tube 2 and so on.22
Experiment 6: Determination of the hydrolyzed sucrose solution byBenedict method1- Place the sugar solution of hydrolyzed sucrose from the previousexperiment in burette.2- Measure 5ml of Benedict quantitative reagent into 100 ml conicalflask add approximately 1g of anhydrous sodium carbonate andfew pieces of porcelain. Heat the mixture vigorously.3- Run in the sugar solution slowly from the burette until a bulkywhite precipitate is formed. Continue the titration by adding thesugar solution drop by drop until the last trace of blue or green hasdisappeared.4- Record the volume of the sugar required to titrate 5 ml of benedictreagent. This volume will be your titer number.Note:1- The end point must be determined while the mixture is still boiling.When the mixture is not boiling atmospheric oxidation occurs andthe green color returns.2- The addition of sodium carbonate to the titration mixture results inthe liberation of CO2, which prevents atmospheric oxidation.3- If the mixture bumps or it becomes too concentrated duringtitration, remove it from the heater, boil 10 ml water in a test tubeand add it to the reaction mixture. Heats the mixture until it boilsagain and continues the titration.4- The tip of the burette must be over the mouth of the flask whilemixture is titrated.23
Calculation:The concentration of the sugar in each tube can be calculated from thefollowing sugar equivalent equationM * V M′ * V′The equivalents for a number of sugars are given as follow, but they onlyapplied if the above conditions are strictly adhered to.25 ml of Benedict’s reagent is equivalent to 50 mg of glucose53 mg of fructose68 mg of lactose74 mg of maltose49 mg of hydrolyzed sugarSince we used 5ml of Benedict reagent which is equivalent to 9.8 mg ofhydrolyzed sugar5ml benedict 9.8 mg5ml benedict Titer no. ml9.8 mg of sugar Titer no. mlX mg of sugar 1 mlX mg of sugar/ ml 1 *9.8 /titer No. *dilution factorDilution factor Final volume / Initial volumeReferences:1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 19782- Harvey,R and Champe,P. Lippincott biochemistry, london.200524
Results SheetFill the table below and plot a relationship between the substrate andproduct concentration.Tube no.[S]mMTiter numberml[P]mg/ml1234525
Results Sheet26
Experiment 7: Estimation of lipase activityLipase is a pancreatic enzyme secreted into the small intestine. Itcatalyses the hydrolysis of triacylglycerols to free fatty acids and glycerolas follow:The release of fatty acids in the solution will cause decrease in the pH andthe rate of the reaction may be followed by:1- Noting the change of pH with time.2- Titration the liberated free fatty acids with standard alkali using asuitable indicator3- By continues titration using an automatic apparatus, (pH-state)which keeps the pH constant and at the same time plots a curve oftiter number against time.Method 2 has been adapted foe this experiment. The liberated free fattyacids at different enzyme concentrations will be titrated with 0.05 NNaOH. Since we are using oils as substrates CaCl2 is used as emulsifyingagent for two reasons:1- to increase the surface area2- To decrease the surface tension, thus the oil drop is effetelyattacked with the enzyme.27
Materials:1- Lipase (1g%)2- Chloroform (10%)3- Fresh oil as the substrate4- Calcium chloride5- Sodium hydroxide 0.05 NProcedurePrepare 6 tubs which contain the Cl2Mix wellD.WLipase(ml)8642010246810-0.1-Incubate in a water bath 37C Enzymeconcentration0.020.040.060.08(µg)Titrate the liberated fatty acids with NaOH noting the time of the titrationshould not exceed 10 min.References:1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 19782- Harvey,R and Champe,P. Lippincott biochemistry, london.20053-Boyer, R. Concepts in biochemistry. John Wiley and sons, New york. 200228
Results SheetDraw a graph of enzyme concentration against ml of NaOH has taken. Isyour curve hyperbolic or liner, comment?Enzyme concentrationml of NaOH (titer no.)29
Experiment 8: Indirect estimation of lactate dehydrogenaseLactic acid produced during anaerobic glycolysis can be converted topyruvic acid with the aid of the enzyme lactate dehydrogenase whenoxygen becomes available. The hydrogen acceptor NAD accepts thehydrogen atoms from the lactic acid and the pyruvic acid moleculeresults. Part of the produced pyruvic acid enters the citric acid cycle afterbeing converted to acetyl CoA. The remainder of the pyruvic acid isconverted into glycogen.LactatedehydrogenaseCH3CHOHCOOHAcetyl CoACitric acid cycleCH3COCOOHGlycogenPyruvic acidIn this experiment, yeast will be used as a source of lactatedehydrogenase. The reaction will be followed by allowing methyleneblue dye to function in place of the natural hydrogen acceptor NAD . Asmethylene blue is reduced it becomes colorless.Lactate dehydrogenaseCH3CHOHCOOHMethylene Blue(blue)CH3COCOOHMethylene Blue(colorless)30
Materials: yeast suspension 5% sodium lactate solution 0.1% methylene blue Water bath 37 C Boiling water bathProcedure:- Label three clean test tubes as a, b and c as followed- Make sure that you shake the bottle of yeast suspension beforeremoving your sampleTest tubeYeastsuspensionSodium lactateMethylene blueA2ml1 dropBC10 drops2mlYeast suspensionPre heated for 10 minin boiling water bathand cooled to 37 Cbefore being used10 drops1 drop1 drop2mlContinue to add methylene blue drop wise ( mixing after each drop)until each solution becomes a uniform light blue in colorMix and place in water bath 37 CObserve the tubes after 10 min. Note any color changes and recordyour observationsReferences:1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 19782- Harvey,R and Champe,P. Lippincott biochemistry, london.20053- Boyer, R. Concepts in biochemistry. John Wiley and sons, New york. 200231
Results SheetExperimentObservationCommentABC32
Experiment 9: Detection of enzymesPepsin: It is protease found in the digestive system of many vertebrates. The pancreas secretes pepsinogen (proenzyme). Activated when chief cells in the stomach release it into HCLwhich activates it. It degrades food proteins into peptides which can be readilyabsorbed by the intestine. Optimum ph 5Detection of pepsin:W.B (37-40 C)1 ml milk 1 ml buffer soln. (pH 5) 0.5 ml pepsinCoagulation occur(10-20 min)Lipase It is a pancreatic enzyme secreted into the small intestine. Catalyses the hydrolysis of triacylglycerols to free fatty acids andglycerol. Optimum ph 8Detection of lipase:1 ml milk 2 drops ph.ph drops of NaOH (0.1 N) till pink color appearW.B (37-40 C) 0.5 ml lipaseTurns colorless(10-20 min)Amylase glycosidase that attack starch randomly. Inactivated by the acidityof the stomach. Optimum ph 733
Detection of amylase:W.B (37-40 C) for 30min1 ml starch 1 ml amylaseTake 1 ml of thesolution every 5 min, and test the presence of starch by iodine solution.Urease Urease is found in bacteria, yeast, and several higher plants. catalyzes the hydrolysis of urea into carbon dioxide and ammoniaurease (NH2)2CO H2O ------------ CO2 2NH3 Optimum ph 7.4Detection of Urease:2.5 ml urea drops Na2CO3 drops phenol red (Red color), then addacetic acid drop by drop until the color change to (Yellow ) filter paperW.B (37-40 C)soaked in ureasered color returns(10-20 min)34
Results SheetExperimentObservationComment35
Experiment 10: Detection of Enzyme mixture36
Results SheetUnknown 1:Contains ExperimentObservationComment37
Results SheetUnknown 2:Contains ExperimentObservationComment38
Results SheetUnknown 3:Contains ExperimentObservationComment39
Results SheetUnknown 4:Contains ExperimentObservationComment40
rate of reaction (enzyme activity) against the different pH at which the enzyme catalytic reactions are incubated. - From the curve, the optimum pH which give the maximum activity of the enzyme will be determined . investigating. 9- Add the lip
Liljeruhm, J. 2019. Exotic Ribosomal Enzymology. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1770. 43 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0567-7. This thesis clarifies intriguing enzymology of the ribosome, the multiRNA/multiprotein
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Methods in Enzymology Volume 148 Plant Cell Membranes EDITED BY Lester Packer Roland Douce DEPARTMENT OF PHYSIOLOGY AND ANATOMY DEPARTEMENT DE RECHERCHE FONDAMENTALE UNIVERSITY OF CALIFORNIA CENTRE D'ETUDES NUCLEA1RES ET UNIVERSITE BERKELEY, CALIFORNIA DE GRENOBLE GRENOBLE, FRANCE ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers
Introduction to Enzymology. Enzymes - Biological catalysts By definition a Catalyst : - Accelerates the rate of chemical reactions . physical methods to solve structure of enzymes Conformation with or without substrate provides functional/biological information Used to identify amino acids involved
Methods in Enzymology Volume LV Biomembranes Part F: Bioenergetics-Oxidative Phosphorylation EDITED BY Sidney Fleischer DEPARTMENT OF MOLECULAR BIOLOGY VANDERBILT UNIVERSITY, NASHVILLE, TENNESSEE Lester Packer MEMBRANE BIOENERGETICS GROUP DEPARTMENT OF PHYSIOLOGY-ANATOMY
Rossmann, Methods in Enzymology 1985, p. 242 Optronics P-1000 film scanner Photo provided by Dieter Schneider, Brookhaven National Laboratory Ron Hamlin - Supper Award talk Diffractometer: automatic but measured only one hkl reflection at a time Robert Sweet, Methods in Enzymology 1985 Ron Hamlin - Supper Award talk
Computational enzymology: modelling the mechanisms of biological catalysts -4- ibuprofen) demonstrate the potential of QM/MM methods to deal with practical questions of drug metabolism22. Cytochrome P450 is an enzyme which is important in drug metabolism. This picture shows QM/MM modelling of cytochrome P450.
References: Methods in Enzymology 100, 468-500 (1983) describes the use of M13 vectors. Methods in Enzymology 154, 329-50. These references and additional references within the volumes describe the original procedures for making mutants. If you know nothing about cloning of genes, use of M13 phage and their life cycle, this is a good place to .
