Over the past five weeks, we have worked on isolating an enzyme and purifying it with different methods every week. For every stage of the enzyme we got, we checked the purity of the enzyme by measuring its enzyme activity and its protein level. For the first week, the enzyme was isolated from the E coli and the extract was placed in a dialysis bag to allow small molecules to leave the bag and to retain the big molecules. Our results for the first week are shown on Table 1 and figure 1 (attached). For the second week, the dialysis product was heated to denature the proteins and then it was precipitated using Ammonium sulfate in order to precipitate the proteins and remove some remaining lipids. Once again the sample was tested for enzyme activity and the rest of the stage was put into another dialysis tube this time to remove the excess ammonium sulfate. The results for week two are show on table 2 and figure 2 (attached). For the third week, we used our stage 3 retentate from the dialysis tube and we run it through a DEAE-Sephacel gel like structure that binds proteins containing anionic groups. At the end we collected the purified enzyme in 20 different tubes and we did an enzyme assay of the 20 tubes and we checked for the tube with the highest absorbance to be our stage 4. Unfortunately our results we got were not very accurate so for day 5 we used another group’s stage 4. We also measured the activity of stage 3 on graph 3 (attached) Table 3: Fraction from the column chromatography and their absorbance value from the spectrophotometer Fraction Absorbance (A410) number Activity detected 1 -0.0698 2 -0.0597 3 -0.0619 4 -0.0703 5 -0.0569 6 -0.0711 7 -0.0386 8 -0.0694 9 -0.0642 10 -0.0698 11 -0.0453 12 -0.0321 13 -0.0444 14 0.0018 15 -0.0226 16 -0.0509 17 -0.0499 18 19 0.0208 No activity(Fraction 18) -0.0285 20 -0.1954 For the fourth week, we used the four different stages we got so far plus the alkaline phosphatase and the low and high protein molecular weight standards to compare the amount of protein on each and also to see if the amount of Alkaline phosphatase increased with each stage. We loaded lane 1 to 7 of our gel and we kept it for one week. On week 5 we took a picture of our gel we run the previous week. We also did an enzyme activity assay to compare the enzyme activity of samples with different concentrations (.25, .125, .0625, .03, and .015) with and without inhibitor. The results were recorded using the spectrophotometer. Our results were not correct for that reason we used Laurie’s values for our plot Table 4: Results of the enzyme activity assay of the enzyme with and without inhibitor (our results) Concentration reaction without in mM inhibitor 0.25 mM 0.125 mM 0.0625 mM 0.03 mM 0.015 mM reaction + 0.1 mM inhibitor 1.7214 1.0806 0.5882 0.2827 0.1446 1.1016 0.4173 0.2792 0.147 Reaction + 0.3 mM inhibitor 0.8879 0.5902 0.2983 0.1822 0.0677 Laurie’s Data [S] (mM) Rate Rate Rate 0.012 0.0528 1/[S] (mM1) 1/v 1/v 1/v (min) (min) (min)w/Pi 0.3mM w/o Pi 0.1mM 83.33 18.93 0.024 0.0801 0.006 0.0044 41.67 12.48 169.49 227 0.05 0.1019 0.009 0.011 20 9.81 112.36 90.9 0.1 0.1201 0.023 0.016 10 8.33 43.1 62.5 0.0337 5 7.85 17.09 30 0.2 0.127 0.06 Figure 4: Line weaver-burk plot of the inverse of the velocity vs the inverse of the concentration of the enzyme (with and without inhibitor) Lineweaver-Burk plot 500 400 1/velocity (min) 300 1/v (min) w/o Pi 200 1/v (min)w/Pi 0.1mM 1/v (min)w/Pi 0.3mM 100 -100 -70 -40 0 -10 y = 5.3003x + 0.999 [0.3mM Pi] 20 -100 50 80 y = 4.1304x + 6.3458 [0.1mM Pi] y = 0.1419x + 6.9413 -200 -300 1/[pNPP] (mM-1) Calculations: 1/V0 =1/Vmax + Km/Vmax (1/[s]) Vmax for graph 1 (inhibitor 0.3 mM) is 1/ 6.941 = 1.001 Vmax graph 2 (inhibitor 0.1 mM) 1/6.3458 = 0.1576 Vmax graph 3 (without inhibitor) 1/0.999 = 0.144 Km is the substrate concentration at which the rate is half Vmax. The slope is Km/Vmax Km(3)= 0.020, Km(2) = 0.6509 Km(1) = 5.306 Calculation of α and Ki At 0.1 α = Km’/Km =0.6509/0.020= 28.3 Ki= [inhibitor]/ [α-1] = 0.1/27.3 = 0.0037 For the 0.3mM inhibitor α = Km’/Km = 5.306/0.020= 265.30 Ki = 0.3/ 264.30 = 0.001 The average Ki = (0.0037 + 0.001) / 2 = 0.002 Table 5: Protein present on our gel and their Rf values Protein Molecular weight Log (m.w.) Rf Myosin 200000 5.3 0.23 B-galactosidase 116250 5.07 0.3 Phosphorylase b 97400 4.99 0.33 Serum albumin 66200 4.82 0.4 Ovalbumin 45000 4.65 0.52 Carbonic Anhydrase 31000 4.49 0.6 Trypsin inhibitor 21500 4.33 0.67 APase ? ? 0.47 Day 1 We did the enzyme isolation, enzyme assay, and dialysis. Fraction Volume ml enzyme assay Total units in fraction mg/ml nanodrop µg/µl=mg/ml Yield Stage 1 8.7 4945.5 43025.85 12.23 Stage 2 11.9 1726.1 20540.59 1.26 136% 0.2 17.6% Stage 3 2.1 Stage 4 3.5 Fraction Rate dA/min Stage 1 0.3297 Stage 2 0.4603 416.25 874.125 0 166% Stage 3 0.111 Day 2 We did the Heat denaturation and ammonium sulfate precipitation and enzyme assay. Day 3 Column chromatography and enzyme assay we searched for stage four out of all the samples we had. Day 4 We did gel electrophoresis to examine the protein composition of the fractions, and an enzyme assay for that we didn’t get a stage 4 we also did the nanodrop. Day 5 We summarized the whole thing and we looked at the gels took pictures of it and we measured the distance between the bands. Concentration reaction without in mM inhibitor 0.25 mM 0.125 mM 0.0625 mM 0.03 mM 0.015 mM reaction + 0.1 mM inhibitor 1.7214 1.0806 0.5882 0.2827 0.1446 1.1016 0.4173 0.2792 0.147 Reaction + 0.3 mM inhibitor 0.8879 0.5902 0.2983 0.1822 0.0677 The purpose of our experiment was to isolate E-coli alakaline phosphatase which is found between the cell wall and the cell membrane of the E-coli. We purified the enzyme week after week using different methods like dialysis, heat denaturation and column chromatography and we measured its purity by checking its activity and also its protein level. The purer the enzyme is, the higher its activity, and the lower its protein level. The first step of the five week experiment was to isolate the enzyme by breaking down the cell wall of the bacteria using lysozyme which hydrolyzes parts of the cell wall and we also added EDTA which removes Ca++ from the cell and DNase that hydrolizes the DNA. After isolating the enzyme (which we called stage 1) we did an enzyme assay and checked the kinetics of the substance. The first results we got showed us. If we look at the overall progression of the enzyme, we can see that its activity has increased over the weeks but also its yield was very different from one experiment to another. The most efficient method to purify the enzyme is supposed to be the one that gives the higher activity and a good yield. The first enzyme assay we performed showed a curve with a slope of 0.3297 and the protein concentration was 12.23 mg/ml. Stage 1 was dialyzed to purify the enzyme and separate it from other molecules. In dialysis, the cellulose bag is semi permeable permiting small molecules to move out and still keeping the big molecule. The activity of the enzyme was low compared to the other weeks’ results and also the protein level in stage one was higher than the other stages because it wasn’t as pure. The product of the dialysis of stage 1 was called stage 2. On the second week we got a big yield 136% of stage 2 because the dialysis caused small molecules to move out and big molecules to stay in. The fact that many of our tubes were placed in the same container may also have had an effect on the exchange of molecules making our yield bigger more than 100%; water molecules moved from one tube to another depending on the osmotic pressure. The result of the enzyme assay showed us much more activity of the enzyme: the slope was 0.4603 and the protein concentration was 1.26 mg/ml. Stage 2 was heat denatured to make the proteins that are still contained in the enzyme insoluble thus separate them more from the enzyme. The alkaline phosphate that is contained in the substance was not affected by the heat because it was still stable at that temperature. After heat denaturating, we used ammonium sulfate which is a salt at different concentrations precipitates proteins of different solubility. That method permits to remove some proteins but it mainly allows the concentration of the enzyme. This method is called “differential precipitation”. The product was put again in the dialysis tube but this time, it was to remove the ammonium sulfate used for the precipitation. The product of that dialysis was stage 3. On the third week, we measured the enzyme kinetics of stage 3 and surprisingly, this time the slope was 0.1110 which is smaller than the value we got for stage 1. However, the protein level was way lower than for the two first stages it was 0.20 which made us assume that the result we got was due to a mistake in the preparation of the enzyme assay or maybe the big decrease in yield (17%). Stage 3 was used for the DEAE-Sephacel column chromatography which is made up of cellulose. The DEAE-cellulose binds to proteins which contain negatively charged groups. It’s important the pH is above the pI of the proteins because the isoelectric pH is when all the sum of all charges is 0. Above the pI, the pH is basic and the net charge of the proteins will be negative allowing them to be displaced by the gel. If the ammonium sulfate was not removed by dialysis, it would hinder the chromatography process because it is a neutral molecule and it wouldn’t have been stopped by the gel. The column fractions that were collected in 20 were used to assay the enzyme activity so that the one with the highest absorbance will be used as the Stage 4 fraction. Unfortunately, we didn’t get a good absorbance for any of our tubes and we had to use other groups’ stage 4. On week four, we took all stages: 1-4 along with low and high molecular weight protein standards and alkaline phosphatase and we run them in a gel. The goal of gel electrophoresis is to separate different proteins depending on their size. The proteins with the lowest molecular weight will run faster in the gel and the biggest protein will be more at the top. Running our stages with different standards helped us see what different type of proteins was present in our stages and also their sizes. The alkaline phosphatase also helped localize the enzyme and see how clear the band was on each stage.