Lab #3: Ion Exchange Chromatography Objective The purpose of this experiment was to separate proteins on the basis of their net charge at a particular pH. In cation exchange chromatography positively charged molecules are attracted to a negatively charged column. Conversely, in anion exchange chromatography, negatively charged molecules are attracted to a positively charged column. Experimental results could be monitored in a predictable way by controlling running pH, salt concentration, and by selecting the type of ion exchanger. Procedure: all procedures are listed in the lab manual.
Results Table 1: Abs 280 Raw Data ABCDE Sample Dilution FactorMeasured Abs280Undiluted Abs 280 (B x C)Graph Bar HEW800. 91873. 44 LOAD100. 8818. 811 Phos 140. 7673. 0682 Phos 210. 5270. 5273 Phos 310. 2480. 2484 Phos 410. 0500. 055 Carb 140. 6462. 5846 Carb 210. 4900. 4907 Carb 310. 1270. 1278 Graph: Abs 280 (Undiluted) Table 2: Biuret Raw Data AFGHIJ Sample Dilution before adding Biuret reagentAdditional dilution factor (Biuret reagent)Total dilution factor into cuvetter (F x G)Measured Abs 540Undiluted Abs 540 (H x I) HEW4052000. 12725. 41 LOAD55250. 1172. 925
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Phos 125100. 1021. 020 Phos 21550. 0730. 365 Phos 31550. 0170. 085 Phos 41550. 0020. 010 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Carb 11550. 0660. 330 Carb 21550. 0150. 075 Carb 31550. 0060. 030 Table 3: Mg Calculations AJKLMNO Sample Undiluted Abs 540Slope from standard curve: Lab 2(abs 540/1mg/ml in cuvette)Undiluted mg/ml (J / K)mlMg proteins of all types (L x M)Subtotals 8x dil HEW mixed with beads 48 Corresponding undiluted original HEW with beads 25. 41 0. 280 90. 750 6 544. 284 LOAD2. 9250. 28010. 30448398. 59 Phos 11. 0200. 803. 64281554. 355 Phos 20. 3650. 2801. 3041519. 56 Phos 30. 0850. 2800. 304154. 56 Phos 40. 0100. 2800. 0357150. 535 477. 6 Carb 10. 3300. 2801. 1791517. 685 Carb 20. 0750. 2800. 268154. 02 Carb 30. 0300. 2800. 107151. 605 23. 31 Table 4: E 1% Calculations ALPQDR Sample of proteinCharge at pH 7Undiluted mg/mlUndiluted g (of protein per liter)% of a liter (1000 gm) Which is proteinUndiluted abs 280E 1% = Abs for a 1 % solutionEstimate of E 1 % HEW90. 75090. 7509. 07573. 448. 092 LOADNeg10. 30410. 3041. 0338. 8108. 528 Carb 1pos1. 17901. 17900. 11792. 58421. 2 Ovomucoid-8. 03. 6 Ovalbumin-8. 06. 2 Ovotransferrin+3. 010. 7 Lysozyme+3. 026. 0 Lab 0 Same as L100% x (P / 1000) Lab 0 Questions: 1. Calculating the concentration of protein of all types in mg/ml in hen egg-white. Total hew mgs used: 544. 91 mgs Total hew ml used : 7 ml Mg/ml = 544. 91mg / 7 ml = 77 mg/ml 2. Comparing the total protein recovered (in mg) from all fractions combined with the total quantity of protein you mixed the beads. Theoretically the total of recovered proteins should be less or sum up to equal the amount first mixed with the beads.
According to table 3: Total recovered mgs= 477. 6. 475 + 23. 31 = 500. 91 mg Total % recovered = (500. 91/ 544. 248) x 100 = 93% 3. Are the data consistent with a hypothesis that the carb#1 fraction has a higher ratio of lysozyme relative to other proteins than the case for the phos #1 fraction? The results in table 3 indicate that Carb #1 has a higher ration of Lysozyme relative to other proteins than the case for the phos #1 fraction, in our case the E1% of the carb 1 was calculated 21. 92, showing that the sample was enriched with Lysozyme. 4.
Are the data consistent with a hypothesis that the carb 1 fraction contains only lysozyme or do the data instead suggest that other proteins also elute with lysozyme in that fraction? In our case the E 1% was recorded to be 21. 92, indicating that the sample for the most part consisted of lysozyme, whose accepted value is E 1% 26. Data also indicates that lysozyme was not the only protein found in carb 1. 5. Present an estimate of lysozyme’s net charge at pH 11. The carb 1 fraction which is mainly constituted of lysozyme is near pH 11 and the net charge of lysozyme is slightly negative. 6. The 200 mM (0. M) Na2 CO3 buffer contains much higher [ ] of Na+ than the 50 mM sodium phosphate buffer. When changing from the phosphate buffer to the carbonate buffer, the difference in Na+ will hinder the desired effect since positive Na+ will compete for binding sited on the negative column. 7. Student’s attempt to make lysozyme bind tighter to CM Sephadex by starting pH lower than 7 is unsuccessful because the charged groups on the column itself are also titratable groups with specific pKa values. At pH 3 the COO ??? group of CM column will become protonated and will no longer are able to bind proteins.
Conclusion Ion exchange columns (beads) work by having a fixed charge on their surface which, before protein added, is neutralized by soluble counterions (like chloride or sodium in our case) from buffers. As previously learned, most proteins contain charged amino acids on their surfaces. Even if proteins have an equal number of positively-charged and negatively-charged amino acids on the surface, they’re never exactly evenly distributed which means that there are areas on the surface of the protein which have an overall positive or negative charge to them.
In our experiment, positively-charged patches on the surface of the protein will be attracted to the CM Sephadex (displacing the sodium counterion in the process). At that point the protein is bound to the surface of the bead. Now examining the possible alternations in pH and their effect on the protein as well as the charge on the column. On the surface of the proteins, the negatively-charged amino acids become protonated, losing their charge. Other groups, such as histidine, which might have been neutral, can now become protonated, picking up a positive charge.
The overall result is that the protein becomes less negatively charged and so will bind more weakly to the column or even detach completely. The exact reverse happens with a negatively-charged resin: proteins bind by virtue of their positive charges on their surface, and as the pH is raised, these groups lose protons and become either neutral or negatively-charged, thus making the protein overall more negative and, once again, allowing it to detach from the resin.