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  1. Analysis and Isolation of Plasminogen Activator from Mammalian CellCulture BrothJohn E. Muñoz Torres and Delaine M. Zayas-Bazán BurgosDr. Vibha Bansal…
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  • 1. Analysis and Isolation of Plasminogen Activator from Mammalian CellCulture BrothJohn E. Muñoz Torres and Delaine M. Zayas-Bazán BurgosDr. Vibha Bansal MentorUniversity of Puerto Rico at CayeyAbstract: Proteins are macromolecules composed of amino acids that are joined by peptide bonds and arepresent in all cells. The importance of having pure proteins is widely known by the scientific community. Itincludes the proteins’ utilization as catalysts, therapeutics, dietary supplements, and in structure studies. Theobjective of this experiment was to use magnetic nanoparticles to separate and ultimately isolate PlasminogenActivator, from a mammalian cell culture. The hypothesis was Plasminogen Activators will be successfullyisolated from a mammalian cell culture broth, HeLa broth. The cell culture was taken from a HeLa cell broth,because of their availability and their immortality. Plasminogen Activator is important in the breaking of bloodclots, an important hemeostatic process. After the isolation of Plasminogen Activator, different methods wereused to analyze it and arrive at results. These methods included a Chromatogram, SDS-Page stained with silverstaining and a Zymography to determine the Enzyme Activity, and Protein Estimation calculations. Theseanalyzes resulted in the proper isolation of Plasminogen Activator at a fold purification of 10. Future studiescould include further analysis of the isolated protein such as crystallography and X-ray diffraction.IntroductionBackgroundroteins, as defined by Hickman(2008), are macromolecules of carbon,hydrogen, oxygen, nitrogen andusually sulfur; composed of chains of amino acidsjoined by peptide bonds; present in all cells. Theword protein is derived from the Greek word‘Proteios’ that means primary. Proteins play themajor roles in all of cellular activities, thereforecontrolling almost all of the activities that composelife itself. Plasminogen is a protein involved in oneof the most important process of Hemostasis, bloodcoagulation. Plasminogen plays a major role inbreaking blood clots. Blood clots are formed whenan injury in a blood vessel is detected. After this aP
  • 2. cascade of enzymes and other biologicalcompounds begin entering the area. These enzymescatalyze the conversion of fibrinogen into fibrincreating the blood clot. Once this blood clot isformed and stops the bleeding the clot needs to bedestroyed. If the blood clot is not destroyed it mighttravel to a smaller vessel causing an aneurism,thrombosis or an embolism. PlasminogenActivators avoid further problems by catalyzingPlasminogen to Plasmin in the presence of a bloodclot. The importance of Plasminogen in essentialbiological processes explains the necessity ofisolating and studying Plasminogen Activators.“Magnetic nanoparticles continue to garnerwidespread interest in biomedical applications, suchas visualization agents in MRI, therapeutic vehiclesfor drug delivery and heat mediators inhyperthermia.” Frimpong and Hilt (2010). Thiswidespread has reached biochemical fields as well,reaching one of the most important processes,protein separation. Pure proteins can be utilized ascatalysts, therapeutics, dietary supplements, forstructural studies and crystallography studies.Protein Separation is achieved through variousprocesses and is of extreme importance to isolateproteins and have them in a pure state. There arevarious processes of isolation cataloged astraditional protein purification proteins. All of thesepresent simple problems: multi-step requiring andlow specificity, meaning that the protein that isseparated is not necessarily the protein of interest.Magnetic Nanoparticles offer simple, economicaland specific protein isolation.Image 1: PABA Treated Magnetic NanoparticlesProblemThe problem of other traditional proteinseparation processes are the complexity of theirsteps, the time consumed, the materials and moneyinvested to acquire small quantities of proteins. Inthis experiment the problem is explored and apossible solution is proposed: the utilization ofMagnetic Nanoparticles in the separation ofproteins. Plasminogen Activators will be isolatedfrom a mammalian cell culture broth, HeLa Broth,and the results will be provided by ProteinEstimation and Enzyme Activity analysis.If the experiment proves to be a solution forthis problem, a change in the traditional methods ofprotein separation can be achieved. In addition tothis, isolating the Plasminogen Activator can be ofgreat help in the studies of the human body,coagulation and the different diseases concerningproper blood coagulation.HypothesisPlasminogen Activators will be successfullyisolated from a mammalian cell culture broth, HeLabroth. The final solution will be a solution of purePlasminogen Activator protein and this will beproven with the calculations and analysis of ProteinEstimation and Enzyme Activity.
  • 3. Materials & MethodsThe experiment was performed in a two partprocess of separation and analysis. The protocolsand materials will be divided by these two divisionsas well.SeparationThe separation is performed with theutilization of Magnetic Nanoparticles alreadysynthesized by the research team of Dr. Bansal.All of these protocols are thoroughly explained inDr. Bansal’s handouts. The first step of theexperiment consisted of labeling the 1.5 mL tubesas follows and placing them in the test tube rack inthe same order: Spent_Group #4, Wash 1_Group#4, Wash 2_Group #4, Wash 3_Group #4, Eluate1_Group #4, Eluate 2_Group #4, and Eluate3_Group #4. The next step was applying thetreatment of PABA to the Magnetic Nanoparticles.This pretreatment of the nanoparticles is done inorder to apply a higher affinity between theMagnetic Nanoparticles and the PlasminogenActivator. This is achieved by adding 4.0 mL ofPABA binding buffer to the centrifuge tubecontaining the previously weighed MNPs. Afteradding the binding buffer sonicate for five minutes,it is immediately placed on the rocker for five moreminutes. Lastly, utilizing a magnet, the supernatantwas decanted. Repeat all of the steps one more time,except sonication of MNP.After the pretreatment, the magneticnanoparticles were ready to bind with the protein.The protein binding procedures were as follows.First we add 1.7 mL of HeLa Broth, and thenlabeled Load_Group#4, to the tube containing theMNPs. The tube is placed on the rocker for thirtyminutes to allow the MNPs to bind the PlasminogenActivator. After this, 1.0 mL of the supernatant istransferred to a 1.5 mL centrifuge tube and thetube was labeled as “Spent”. Lastly, wemagnetically decanted the rest of the Spent into thewaste container.Once the proteins had bound to the particles,the washing procedures took place. Firstly, 4.0mL of Binding buffer is added to the tube andplaced on the rocker for five minutes. Immediatelyafter, 1.0 mL of the wash is collected in tubelabeled Wash1_Group#4. Lastly, the rest of theBinding Buffer is magnetically decanted. Thesesteps are repeated two more times.The last procedure of the separation processis the elution. At this point, only our protein ofinterest was in the solution bound to the MNP. Theelution process is performed in order to separate theMNP from the Plasminogen Activator. The processbegins by adding 1.0 mL of PABA Elution buffer tothe MNP. Then the tube containing the MNPs isplaced on the rocker for five minutes. After eachelution step, 1.0 mL of the supernatant is collectedin a 1.5 mL centrifuge tube, labeledEluate1_Group#4. The process is finished bymagnetically decanting the supernatant if there issome left. These steps had to be repeated two moretimes.
  • 4. Image 2: Brief summary of the Separation ProcessSample AnalysisIn order to prove the hypothesis,further steps need to be completed. These include aChromatogram. The Chromatogram is performed bytaking a microwell plate and adding 200 μL of eachsample to individual wells. Later the absorbencieswere read at 280nm and the Chromatogram isprepared.The next procedure completed inorder to further analyze our samples is the SDS-Page. The following reagents are mixed in order topolymerize the separation gel at 10.0%: 1.68 mL ofdH2O, 1.67 mL of Separating buffer, 2.22 mL ofAcrylamide, 50.0 µL of 20% SDS, 100.0 µL of10% Glycerol, 1.67 µL of TEMED, and 50.0 µL ofAPS (100mg/ml). The following reagents are mixedin order to polymerize the staking gel at 4.0%: 1.25mL of stacking buffer, 0.662 mL of Acrylamide,25.0 µL of 20% SDS, 25.0 µL of 10% Glycerol,6.25 µL of TEMED and 25.0 µL of APS (100mg/ml). After polymerizing the gels the samples areheated at 80°C for ten minutes and immediatelyplaced on ice. This is done in order to furtherdenaturalize the proteins. The samples is preparedutilizing standard SDS Sample Preparation andloaded in this order: Lane 1Ladder, Lane 2Load, Lane 3 Spent, and Lane 4 Eluate. Afterthe gel is successfully run, it is incubated over nightin the rocker on fixative solution (methanol, aceticacid and dH2O). It is then submitted to a series ofwashes in Ethanol 10%, Farmers Reagent, SilverNitrate, Sodium Carbonate, Distilled Water andDeveloper.A Zymography Gel is polymerized in orderto obtain the Enzyme Activity of the samples."Zymography is an electrophoretic technique usedto identify proteolytic activity in enzymes separatedin polyacrylamide gels under nonreducingconditions” (Kleiner and Stetlerstevenson, 1994).In order to polymerize the separating gel 10.00%the following reagents are mixed: 1.788 mL ofdH2O, 1.67 µL of Separating buffer, 2.22 µL ofAcrylamide, 50.0 µL of 20% SDS, 100.0 µL of10% Glycerol, 50.0 µL of Fibrinogen, 40.0µL of Plasminogen, 1.67 µL of TEMED, 2.0 µLThrombin, and 50.0µL of APS (100mg/mL). Thefollowing reagents are mixed in order to polymerizethe stacking gel at 4.00%: 2.975 mL of dH2O, 1.25mL of Stacking buffer, 0.662 mL of Acrylamide,25.0 µL of 20% SDS, 25.0 µL of 10% Glycerol,0.25 µL of TEMED, and 25 .0 µL of APS(100mg/mL). The samples are not denaturalized ascustomary in SDS-Page and were loaded in thesame order as the SDS-Page had been loaded. :Lane 1Ladder, Lane 2 Load, Lane 3 Spent,and Lane 4 Eluate. The Zymo gel runs at atemperature of 4°C, this is purposely done in order
  • 5. to forbid the enzyme from digesting the substratewhile running. Once the gel is successfully run, it isthen incubated at 37°C for eight hours in 1xincubating buffer. After eight hours of incubation,the gel is stained for twenty- five minutes inCoomasie Blue and distained extensively.The final step of our Sample Analysis wasthe Protein Estimation and the Enzyme Activity.The Protein Estimation is performed with theobjective of learning the protein concentration ineach sample. It was performed utilizing theBicinchoninic Acid Method. A purple coloredreaction product is formed by the combination oftwo molecules of BCA with one cuprous ion. Themore protein available resulted in a deeper purplecolor.. In a microwell plate BCA and Copper areadded to the sample and then incubated at 36°C.Since the color was purple, the absorbance wasmeasured at 562 nm. The Enzyme Activity wasperformed utilizing the Chromogenic Assay. It wasalso placed in a microwell plate, but it was notincubated. When the enzyme breaks up thesubstrate, a yellow color is formed. Because of thisyellow color the absorbance is read at 405nm.ResultsAll of the different experimentations weresuccessful. This success was measured followingdifferent procedures, including the Chromatogram,Protein Estimation and Enzyme Activity analyses.The protein was isolated at a fold purification of 10.The first analysis of our separation samples was theChromatogram, (see Graph 1 and Table 1) TheChromatogram shows the diverse absorbencies ofour samples at 280 nm. The absorbance shows thequantity of protein in the diverse samples. Note thatthe Eluate samples contain the least amount ofprotein, proving that our experiment was successful.From the bulk of proteins, the smaller amounts ofour protein of interest were isolated.Graph 1: Chromatograph showing thediverse absorbencies at 280 nmTable 1: Corresponding quantities of thechromatography analysis0.0000.5001.0001.5002.0002.5003.0000 2 4 6 8 10 12 14 16 18AbsotbanceVolumeChromatogramEluateWashLoaVolume TotalAbsorbanceNetAbsorbanceBlanks1.7 2.926 2.864 BindingBuffer3.4 2.803 2.741 0.0627.4 0.359 0.29711.4 0.130 0.06815.4 0.106 0.04416.4 0.143 0.074 ElutionBuffer17.4 0.238 0.169 0.06918.4 0.199 0.130
  • 6. Image 3: SDS-Page with Silver Staining Image 4: SDS-Page with Silver Stainingwith accurate protein quantitiesGraph 2: Protein EstimationProtein Estimation was performed in order to learnthe concentration of protein available in ourseparation samples (see graph 2, table 2, images 3and 4). This estimation also showed the success ofthe micropipetting since the variable showing theaccuracy of the results is fairly close to 1, which isthe standard of lineal regressions. Please note thatonce again the least amount of protein is found atthe Eluate Samples. This is because of the reasonsmentioned before: the eluate contains only theprotein of interest therefore there is less protein.The other proteins are no longer available in they = 1.4221x - 0.0141R² = 0.993-0.2000.0000.2000.4000.6000.8001.0001.2000.000 0.200 0.400 0.600 0.800AmountofProteinAbsorbancyat 562nmProtein EstimationLadderProtein of interestPA approximatemolecular weight 75HeLaLoadHeLaSpentEluate Ladder HeLaLoadHeLaSpentEluateProtein of interestPA approximatemolecular weight 75
  • 7. sample. The lineal regression also helped in theSDS-Page Gel. The first one (see image 3) wasperformed without any protein estimation and theoverlapping bands prove so. The second one (seeimage 4) was performed after the protein estimationand the gel has a cleaner and simpler look, and itsbands are clearly separated.Table 2: Protein Estimation AssayWell Sample Sample Volume (µl) dH2O(µl) BCA (µl) Absorbance Final Protein Concentration(Average)F1 Blank - 11.5 169 0.065F2 BSA Standard x 2.3 9.2 169 0.254F3 BSA Standard x 6.9 4.6 169 0.436F4 BSA Standard x 11.5 - 169 0.773F5 BSA Standard x/10 2.3 9.2 169 0.096F6 BSA Standard x/10 6.9 4.6 169 0.078F7 BSA Standard x/10 11.5 - 169 0.158F8 BSA Standard x/100 2.3 9.2 169 0.073F9 BSA Standard x/100 6.9 4.6 169 0.081F10 BSA Standard x/100 11.5 - 169 0.080F11 HeLa Load x/10 2.5 9.0 169 0.097 1.55F12 HeLa Load x/10 11.5 - 169 0.191G1 HeLa Spent x/10 2.5 9.0 169 0.228 1.47G2 HeLa Spent x/10 11.5 - 169 0.610G3 HeLa Eluate 11.5 - 169 0.096 0.03G4 HeLa Eluate 11.5 - 169 0.095The Enzyme Activity Analysis (see graph 3and table 3) was performed in order to learn thespecific activity of protein in our separationsamples. The analysis also provides anascertainment that the protein isolated was thecorrect protein. We utilized the Zymol Gel in orderto create a protein clot. The gel had proteins all overand the protein of interest, Plasminogen Activator,only in one of the lanes. The lane containing theprotein shows a white spot, which suggests that thePlasminogen Activator was breaking the clot. Thesuccess of the micropipetting since the variableshowing the accuracy of the results is fairly close to1, which is the standard of lineal regressions.
  • 8. Table 3: Enzyme Activity Assay Image 5: Zumography gel with Coomasie Blue StainingWell Sample Tris/ NaCl/ TweenReaction Buffer (µl)D-VLKX/10(µl)Plasminogenx/10(µl)SampleQuantity(µl)Absorbance ActivityIU/mlA1 Blank 120 30 30 0 .073A2 Blank Media 70 30 30 50 .147A3 Blank Media 20 30 30 100 .168tPA x/20A4 A 117.5 30 30 2.5 .097 34.72A5 A Duplicate 117.5 30 30 2.5 .106A6 B 115 30 30 5 .124 69.44A7 B Duplicate 115 30 30 5 .108A8 C 110 30 30 10 .252 0.00A9 C Duplicate 110 30 30 10 .172A10 D 100 30 30 20 .352 277.78A11 D Duplicate 100 30 30 20 .293tPA x/200A12 A 117.5 30 30 2.5 .090 3.47B1 A Duplicate 117.5 30 30 2.5 .090B2 B 115 30 30 5 .098 6.94B3 B Duplicate 115 30 30 5 .089B4 C 110 30 30 10 .094 13.89B5 C Duplicate 110 30 30 10 .099B6 D 100 30 30 20 .122 27.78B7 D Duplicate 100 30 30 20 .123B8 Broth 20 30 30 100 .187 150.32B9 Spent 20 30 30 100 .749 68.68B10 Eluate 20 30 30 100 .099 1574.47EluateHeLaSpentHeLaLoadProtein of interestPA approximatemolecular weight 75
  • 9. Graph 3: Results and Linear Regression of the Enzyme Activity of PADiscussionThese results prove our initial hypothesis:Magnetic Nanoparticle Separation is a viable andsuccessful method of separating proteins. Duringour experimentations our hypothesis was provenand is sustained by our results. Further comparisonsshould be conducted in order to further prove theseparation success of magnetical nanoparticles.Various works such as the work conducted byDamira (2010) showed the efficiency of separationbased on Magnetic Nanoparticles. They utilizedsimilar procedures of separation in order to separateListeria monocytogenes from food matricesFurther works should include also furtheranalysis of the protein isolated: PlasminogenActivator. These may include crystallography andx-ray diffraction. This protein is of extremeimportance in various fields of biomedicine andthese studies might help find a cure to coagulatingdiseases.“Magnetic nanoparticles continue to garnerwidespread interest in biomedical applications, suchas visualization agents in MRI, therapeutic vehiclesfor drug delivery, and heat mediators inhyperthermia” (Reynolds and Hilt ,2010) .MagneticNanoparticles have spread throughout variousbiomedical fields. These experimentationsconcerning protein separation based on magneticnanoparticles are just some of the various stepsneeded. Science is moving forward, and Medicine istagging along. These just provide another windowof opportunity to find better treatments, therapies,diagnostic tools and prognosis tools. Having suchtechnology can change the medical world. Ourexperiment forms part of the smaller picture; oursuccess just adds to the bigger picture another color.AcknowledgementsThis work was funded by the RISE Programat the University Puerto Rico at Cayey and by they = 1177.9x - 5.0193R² = 0.94780.0050.00100.00150.00200.00250.00300.00350.000.000 0.050 0.100 0.150 0.200 0.250 0.300Enzymeactivity(IU/mL)Abs at 405 nmEnzyme Activity
  • 10. Investigation Laboratory of the ChemistryDepartment at the University of Puerto Rico atCayey. We gratefully acknowledge Dr. V. Bansalfor her assistance in the experimentations andprocedures. Without Dr. V. Bansal’s assistance theexperimentation would have not been possible. Wealso dearly thank Mr. Carlos Castrodad, Mrs.Natalia Espada and Mr. Javier Rosado, ourlaboratory technicians throughout the experiment.We thank all of the staff of the Laboratory and ofthe RISE Program.Literature CitedBalaure P, Buteica A, Grumezescu A, Mihaiescu D,Mihaiescu O, Mogosanu D, Trăistaru V, Vasile B.2012. Bioassay and Electrochemical Evaluation OfControlled Release Behavior Of CephalosporinsFrom Magnetic Nanoparticles Digest. Journal ofNanomaterials and Biostructures. [Internet];[Revised 2012 February 24, Cited 2013 May 16]7(1). <ahref="http://search.ebscohost.com/login.aspx?direct=true&db=a9h&AN=74616350&site=ehost-live">BIOASSAY AND ELECTROCHEMICALEVALUATION OF CONTROLLED RELEASEBEHAVIOR OF CEPHALOSPORINS FROMMAGNETIC NANOPARTICLES.</aChandler WL, Ferrell C, Kha H, Lee J, Tun T.2003. Comparison of Three Methods for MeasuringFactor VII Levels in Plasma. American Society forClinical Pathology. [Internet]; [Cited 2013 May 18]12(34-39). 4 DOI: 10.1309/C8T8YNB4G3W45PRFhttp://ajcp.ascpjournals.org/content/120/1/34.full.pdfEisenhour David, Hickman Jr. Cleveland, I’AnsonHelen, Keen Susan L, Larson Allan, Roberts LarryS. 2008. Integrated Principals of Zoology.Fourteenth Edition. United States of America.McGraw-HillErf Gisela F, Kanayeva Damira A, LiYanbin, Rhoads Douglas, Slavik Michael F, TungSteve, Wang Ronghui. 2012. Efficient Separationand Sensitive Detection of Listeria monocytogenesUsing an Impedance Immunosensor Based onMagnetic Nanoparticles, a Microfl
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