Purification of Alcohol Dehydrogenase From Bovine Liver

catharsis of alcohol Dehydrogenase From bovid LiverJekathjenani Ratnakumaran Namrata VermaIntroductionIn the world of chemistry, there argon millions of enzymes, merely in this lab the enzyme collapse is bovine intoxicant dehydrogenase. This enzyme occurs in variant mammalian tissues, simply generally found in high concentrations in the organs much(prenominal) as liver and kidney. jibe to its name bovid alcohol dehydrogenase, which implicates that it is collectively formed from bovine (cow), alcohol and the enzyme dehydrogenase. The protein was extracted from the liver of bovine. Alcohol is an radical compound which contains carbon atom (single bonds) and hydrogen atoms. This alcohol is available in various forms of liquid and used for a variety of purposes. According to its properties, alcohol is a hydroxyl group which has a sweet olfactory perception similar to fruit. Alcohol be further divided and identified into different groups and also they ar polar. As they p ossess hydrogen bonding they wear higher boiling points. Dehydrogenase is a type of an enzyme which oxidizes a substratum by a reduction reaction that transfers one or more hydrogen H- to the electron acceptor which is nicotinamide adenine dinucleotide+ /nicotinamide adenine dinucleotideP (nicotinamide adenine dinucleotide) or FAD Flavin coenzyme (Shibusawa et al, 2004). Collectively, it forms alcohol dehydrogenase, which is vasopressin persuaded by ethanol and acetaldehyde as they relate to carbon catabolite repression. It is also zinc containing enzyme which is activated by glutathione and EDTA, which contains heavy metals (Pateman et al, 1983). Many organisms contain an alcohol dehydrogenase enzyme which catalyzes the NADPH dependent of aromatic and aliphatic aldehydes into subsequent alcohols and catalyze the reduction of glyceraldehyde to glycerol (Arslanian et al, 1971). Alcohol dehydrogenase contains a several isozymes which catalyze the oxidation of primary and secondary al cohols to convert into aldehydes and ketones (Arslanian et al, 1971). The molecular(a) incubus of this enzyme is 39677.13 Da and it is do up of 374 amino acid sequence. The monoisotopic peck of this enzyme is 39651.32 and its pH value ranges between 8.6 to 9.0 with an extension coefficient of 12.6 and an isoelectric point at 5.4, its theoretical pI is 7.46.The alcohol dehydrogenase is also known for its battle against alcohol , its toxic molecules which negotiates with the nervous dodging so, the body organs which consist of high toxic of alcohol atomic number 18 liver and stomach which converts alcohol to acetaldehyde which is even more toxic substance and further it is conversted to acetate which is utilized by the cells render within our body (Goodsell et al, 2001). So boilers suit, alcohol dehydrogenase converts potentially dangerous molecule into food ustilized by the cells preent within the body.In human bidy, alcohol dehydrogenase can clear upto nine different kinds of alcohol dehydrogenase each having different properties. For practice session in liver beta3 enzyme(Goodsell et al, 2001).These each enzyme is formed of cardinal subunits and they can be mixed and match to create mixed dimerss which are more active.Alcohol dehdrogenates also modifies certain other alcohols with giving outcome of dangerous products such as methanol. These by products are converted into formaldehyde by help of alcohol dehydrogenase (Goodsell et al, 2001).Catalytic bodily function of alcohol dehydrogenaseNADPH + an aldehyde NADP+ + an alcoholMethodsIn order to conduct this laboratory experiment, All the need mechanism and materials were provided during the lab. Certain precautions and safety rules were followed such as gloves, safety glasses and lab coat. This lab was conducted for about season of 11-12 weeks. According to the article (Arslanian et al, 1971) most of the shouts and procedure was followed. Purification of the enzyme was carried out by followin g up eight footfalls. Precautions were made turn the experiment was performed. Equipments were rinsed with distilled water before starting the experiment. The reagent and cowcatcher beginning were brisk with distilled water. All procedures were carried out at 0-40C.Buffer Preparation The first step heterogeneous in the pilot light preparation. The stock solution, 0.1M Tris HCl at pH 7.6 was prepared by dissolving 12.14 g of Tris base with 1000 ml of distilled water and the pH was adjusted to 7.6 by adding diluted HCl. The Tris HCl buffer solution with different concentrations such as 10mM (pH 7.5), 40mM (pH 7.6) and 50mM (pH 7.5) were prepared by diluting the stock solution with distilled water. Sodium Chloride elutant buffer (0.16M NaCl) was prepared by dissolving 9.3504 g of NaCl with 1000 ml of distilled water.Preparation of Homogenate The second step involved the preparation of the homogenate. The bovine liver was match in a Waring blender in 90 ml of 0.32 M sucrose in 1 0mM Tris HCl buffer at pH 7.5. Approximately 27.39 g of sucrose was added in 250 ml of 0.01M Tris HCl to make 0.32M sucrose in 10mM of Tris HCl at pH 7.5. The homogenate was centrifuged at 15000 RPM for 30 minutes using centrifuge-Sorvall RC5 refrigerated centrifuge SS 34. ammonium ion Sulfate Fractionation Step three involved ammonium sulfate fragmentation. The homogenate was 35% saturated and equilibrated with ammonium sulfate by dissolving 20.9 g of ammonium sulfate in 250 ml of distilled water. The supernatant was centrifuged at 15000 RPM for 30 minutes and the precipitate was discarded. Then, concentration was increase to 60% saturated ammonium sulfate by dissolving 16.4 g in 500 ml of distilled water. The suspension was centrifuged at 15000 RPM for 30 minutes. The obtained gray pellet was dissolved in 40mM Tris HCl buffer at pH 7.6. Then, the solution was dialyzed against 2L of 0.04M Tris HCl at pH 7.6 for 24 hours and again dialyzed with same buffer for a nonher 24 hours. p layacting DEAE-Sepharose Chromatography The fourth step involved DEAE-Sephrose Chromatography. DEAE-Sepharose editorial that can hold up to 10ml volume was applied. The column was equilibrated by applying four magazines of 10ml of 40mM of Tris HCl, pH 7.6 buffer. About 10ml volume of centrifuged and dialyzed material was applied through the column. The column was washed with the same buffer (40mM of Tris HCl, pH 7.6) and then eluted by 40mM of Tris HCl with 50mM of NaCl. About 1 ml volume of twenty fractions of enzyme solution was collected using microfuge tubes.Enzyme Activity Assay Fifth step involved measuring enzyme bodily function using a spectrometer. The enzymatic activity was initiated with 1mL of volume of blank solution containing 20 l of distilled water, 10 l 33mM of ethanol, 10 l of 0.26mM of NAD+ and 960 l of 0.1M of glycine buffer. Enzymatic curb activity was measured by taking heart volume of 1000 l containing 20l of enzyme solution, 10 l 33mM of ethanol, 10 l of 0.26mM of NAD+ and 960 l of 0.1M of glycine buffer. The wavelength was set up at 340nm and measured using Cary 50s and 60s spectrometer. One unit of activity equals 1mol Nvasopressin produced per min based on the absorption coefficient of 6220 mol/l/cm for NADH at 340 nm. The above procedure was repeated for kinetic analysis and the range of ethanol concentration used was 20 to 25 mM. The observed data were fitted using Lineweaver -Burk kinetic plots.Gel filtration The sixth step involved gel filtration. The enzyme was precipitated by 62% saturated ammonium sulfate and dissolve in 10ml of 50 M Tris-HCl, pH 7.5. The suspension was centrifuged for 20 min at 10000 RPM. Then, the column of Sephadex G-50 was run with 10 ml of enzyme solution. The column was equilibrated and washed with 50 M Tris- HCl buffer, pH 7.5. Then, the column was eluted by 50mM of Tris HCl with 50mM of NaCl. Around 10 fractions were collected at the rate of 1 ml/min in a microfuge tube. The highest highest specif ic activity fractions were precipitated by 62% saturation with ammonium sulfate. In the final step, the enzyme was redissolved in 5 ml of same buffer and apply to the column of Sephadex G-50 under the same conditions. Again, the highest specific activity fractions were precipitated by 62% saturation with ammonium sulfate.Performing CM-Sephadex chromatography The precipitated enzyme was dissolved in 1 ml of potassium phosphate buffer contain 0.02M of NaCl, pH 7.0 and the enzyme solution was dialyzed against the same buffer for 2 hours. 10ml of non diffusible material was applied to a CM-Sephadex column. Then, the column was equilibrated with the same buffer. Then, the enzyme was eluted from the column with two column volumes of 0.16 M of NaCl (20ml). 10 fractions were collected and precipitated with 62% saturated ammonium sulfate.Bradford Assay The eighth step involved Bradford Assay. The data (absorbance) observed from Bradford Assay Standards was used for calculating the spile of BSA in g. The final step used in this experiment was SDS PAGE method. About 20l of enzyme with loading buffer was rigorous on the gel and by observing the gel, the mass of the protein was calculated.ResultsBovine Alcohol Dehydrogenase (ADH) was purified by following up few methods. The experimental results were observed and recorded for appropriate methods. Using DEAE Sepharose Chromatography, fractions were collected and all the fractions were appeared colorless. The enzymatic assay activity was measured at 340nm using spectrometer. The cast 1 indicates that the enzyme activity was increased by take up the NADH. The highest specific activity was selected based on the graph obtained in the enzyme kinetic activity. However, this method failed, resulting no increased activity. The enzyme kinetic activity had done for all the fractions, but none of them shown the accurate result. The graph obtained from the spectrometer does non show the increased activity of the enzyme to conclude the presence of protein. The result of the enzyme activities of collecting fractions was shown in systema skeletale 5, 6, 7, 8 and 9 respectively. However, the Bradford assay method was performed and the absorbance of the steps and the enzyme were recorded in the following tables. Based on these values, the graph of standard curve of absorbance versus mass of BSA was plotted. put off 1 The following table represents the recorded values of absorbance at 540 nm and calculated the mass of the BSA using the Bradford assay method inscribe 1 Represents the enzymatic activity obtained from the purified protein ADH after ammonium fractionation method had performed and the peaks are showing that activity is increased. The above plot was obtained at 340 nm using Cary 50s-60s spectrometer and it was run as two parts for 4 minutes.Figure 2 Represents the standard curve of A595 versus mass of ADH protein obtained from the Bradford assay method. From the slope value obtained from the curve, th e mass of the ADH protein was calculated.The mass of the protein calculated from the figure 2 is 4.766 g and concentration of the protein is 4.766 g / 25 lSDS-PAGE methodTable 2 Represents the recorded values of SDS-PAGE method for the determination of molecular weight of the ADH purified proteinProteinMolecular weight (Dalton)Log (Molecular Weight)Mobility (cm) mountain range 1600004.7784.8Strand 2500004.6995.1Strand 3400004.6026.5Strand 4250004.3987.0Strand 5200004.3019.3Figure 3 Represents the SDS-PAGE analysis of purified protein bovine Alcohol Dehydrogenase (ADH). The graph was plotted with log of molecular weight versus mobility of protein based on the SDS-PAGE values.Figure 4 Represents the single band on an SDS-PAGE gel (9th lane). This figure shown the proof of the protein ADH present in the enzyme solution and mass of the protein was calculated based on the obtained SDS-PAGE results.From the figure 3 and 4, the mass of the protein calculated is 345143.74 DaFigure 5 Represe nts the enzymatic activity obtained in the fraction 9th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer.Figure 6 Represents the enzymatic activity obtained in the fraction 10th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer.Figure 7 Represents the enzymatic activity obtained in the fraction 12th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer.Figure 8 Represents the enzymatic activity obtained in the fraction 13th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer.Figure 9 Represents the enzymatic activity obtained in the fraction 14th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer.DiscussionAccording to the experimental study, the outcome results were not satisfying, so overall the experiment was not winnerful it failed. Based on the SDS-PAGE , ADH purified protein was not much visible intelligibly on the gel. Proteins are viewed as bands. SDS-PAGE results indicates that smaller protein molecules are at the bottom of the gel and larger molecules are at the top of the gel. This is showing that SDS-PAGE gel divorce the protein molecules based on the size and mass of the protein. approximately of the protein bands are viewed in between the molecular weight, 100 kDa and 30 kDa. Determining mass and purifying the protein, Bovine Alcohol Dehydrogenase using the Bradford assay and SDS-PAGE procedure was conducted successfully using this experiment. The result obtained in the SDS-PAGE and Bradford Assay are differ from the standard value and the concentration of the protein was determined using these methods. Based on the molecular mass on the EXPASY website, the standard molecular mass of the ADH protein is 39677.13 Da. The experimental mass of the ADH protein is 345143.74 Da. The mass battle is a large number. This could o ccur due to the experimental errors. The experimental errors can be avoided by use equipments and following the instructions in a proper manner. Predicting the protein band on SDS-PAGE gel could cause the error. Moreover, the purgation method such as DEAE Sepharose Chromatography was performed to test the enzyme activity of the protein. The obtained results are shown as a figure 5, 6, 7, 8 and 9 in the results section. The overall results obtained in these figures indicated that the experiment was not turned successful. The figure 5, 6, 7, 8 and 9 shown that enzyme activities are decreasing and wiggling. They are not constantly increasing or decreasing. Therefore, it was concluded that the purification of the enzyme was not turned positive and it could be due to the human errors occurred while conducting the experiment. This could be possible due to various reasons such as, during measurements for making the solution at the very beginning may be the concentration required was not appropriate, due to human error it was not properly mixed. It could also be possible that while grinding the liver , certain chunks of the liver were still not properly collected due to which the totality of liver used was not effective to obtain supportive and positive results.The variability of the results presented here is firing of certain atoms during the process of purification as their was no enzyme activity observed.The substrate studies of the alcohol dehydrogenase isolated from the bovine liver have demonstrated the hydrophobic site for binding alcohol (Arslanian et al, 1971). The article mentioned that the buffer that has a low ionic strength is used for the enzyme adsorbtion which caused the incomplete deactivation of enzymes and it was proven evidently (Arslanian et al, 1971). Moreover, as there is no enzyme activity measured in step 5 (DEAE- Sephrose Chromatography), the gel filtration and CM-Sephadex Chromatography method was not performed for this study. The enzym e purification might get succeeded if the study has performed these two methods. The article mentioned that gel filtrations on Sephadex G-100 has successive ability to separate the enzyme from non enzyme protein (Arslanian et al, 1971.)For further studies, more information is required before conducting the study as well as the time allotted was less, due to which it could suggest certain results and test were not done at the appropriate time. In conclusion, the study was conducted by following the method listed in the article. This studys report discussed the properties and successful method for the purification of enzyme, Bovine Alcohol Dehydrogenase. Even though article procedures were followed, errors occurred which resulted in deviations in results. However, the methods of Gel filtration and CM Sephadex Chromatography where successive but could not be conducted in this lab because the enzyme activity was limited after DEAE Chromatography was performed. More caution should have g ained while conducting the experiment. It is emphasized that further research on enzyme purification method could improve the results and find success in the study.AppendixSample calculation 1Volume of one microliter= 0.001mLVolume of 20 microliter= (0.001 ml x 20 L) / 1 L= 0.02 mlTherefore, mass of protein in 1mL of stock solution= 0.10 mgMass of protein in 0.02 ml of stock solution = (0.10 mg x 0.02 ml) / 1 ml= 2 x 10-3 mgTo convert mg to L, multiply by 1000,Mass of protein= 2 x 10-3 x 1000= 2 gAbsorbance of the ADH purified Protein, y = 0.2544Slope Line of equation Y=mx+bY= 0.0505 x + 0.01370.2544 = 0.0505 x + 0.0137The mass of the protein, x = (0.2544 0.0137) /0.0505 g= 4.766 gConcentration of the protein, C = mass/ volume= 4.766 g / 25 l= 0.19 g/ lTotal mass that acquire= Conc. X Total volume= 0.19 x 1000 l= 190.64 gSDS- PAGE methodAbsorbance of the ADH purified Protein, y = 0.2544Slope Line of equation Y= mx+bY= 6.0902 x + 33.9820.2544= 6.0902 x + 33.982X= (0.2544 33.982) / 6.0902= 5.538The mass of the protein = 105.538= 345143.74 DaReferencesArslanian,M.J., Pascoe,E,. and Reinhold,J.G., (1971) Rat Liver Alcohol Dehydrogenase.Dept. of Biochem.School of Medicine, American University of Beirut.125,1039-1047.Alcohol Dehydrogenase(ADH)The university of Minnesota Biocatalysis/Biodegradation Database.Calzyme. Lab.inc.Shibusawa,Y.,Fujiwara,T.,Shindo,H., andIto,Y. (2004) Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography, J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci.799(2)239-44.Pateman,J.A., Doy,C.H.,Olsen,J.E.,Norris,U., Creaser. E.H., and Hynes,M.(1983) Regulation of Alcohol Dehydrogenase (ADH) and Aldehyde Dehydrogenase (ALDDH) in Aspergillus nidulans.Proceedings of the Royal society.Bio.Sci.217, 243-264.Ward,W.W., and Swiatek,G.,(2009) Protein purification.The state University,, Scool of Environmental and Biology Science,Department of Biochem. And Microbio.76,1- 21.Goodsel l,D.(2001) Alcohol Dehydrogenase.Molecule if the month. RCSB.Protein Data Bank.doi 10.2210.