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THEORY OF PRACTICAL Questions 52 to 57 refer to the following information. You have been given a mixture of 5 proteins (A - E) to separate and identify. You have been told that some of these proteins are coloured, containing a heme prosthetic group, which absorbs strongly at 410 nm. Two of the proteins contain 2 identical subunits (equal molecular weight). You have carried out the following experiments: Experiment 1. An aliquot of the mixture (A-E) was loaded onto DEAE cellulose at pH 7.0. One mL fractions were collected and the absorbance measured at 410 nm and 280 nm. The first peak eluted contained A and B. A second peak, eluted after the addition of 0.5 M NaCl at pH 7.0 contained two proteins, C, D and E. three Absorbance Cathode -ve 1.8 1.6- 1.4 1.2 1.0 0.8 0.6 0.4- 0.2- 0 Bution Profile: DEAE cellulose pH 7.0 Peak 2 0.000²080 15 μL used in Experiment 2 Peak 1 well 5 4 0.5 M NaCl Experiment 2. A 15 μl sample of Peak 1 from experiment 1 (proteins A&B) was loaded onto a 1.5 % agarose gel, made up in Tris barbitol buffer, pH 8.8. The sample was subjected to electrophoresis at 60 V for 45 min and the gel was then stained with Coomassie blue for 40 min. Destaining followed until the background was clear. The resultant destained gel is shown below. A 10 15 Volume (ml) A 280 nm -0-A 410 nm 20 Anode +ve migho sd

52. What is the most likely molecular weight of protein A? 53. 54. A. B. C. D. E. What is the pl of protein A? A. B. C. D. E. 15 000 30 000 45 000 70 000 90 000 A. B. C. D. E. 10.5 9.5 8.4 6.5 4 One of the proteins in your mixture (A - E) is a histone H2A. Which protein (A - E) is Histone H2A? Protein A Protein B Protein C Protein D Protein E thiw zashuoloo ei nitong aidTnimudlevo anisini Y Trimudisvo ai (3-A) niston doirW 000 24-10 trigiow

Experiment 3. Another aliquot of the protein mixture (A-E) was loaded onto DEAE cellulose at pH 5.0 using the same procedure as in experiment 1. The first peak eluted contained three proteins A, B and D. The second peak, C and E, was eluted after increasing the ionic strength of the buffer. Experiment 4. Another aliquot of the protein mixture (A-E) was loaded onto a G-50 Sephadex column (cut-off >50 000) equilibrated with 50 mM Tris pH 7.5. One mL fractions were collected after measuring a 30 ml void volume. Two protein peaks were eluted. The first peak contained one protein, A. Absorbance 1.0 0.8 0.7- 0.5 M 0.6 NaCl W 0.5 0.4 0.3- 0.2- 0.1- 0.0000 5 Absorbance 0.9 3.0 2.5- 2.0 1.5 0 0.5 Elution profile: DEAE cellulose pH 5.0 1.00x4 0 0.00-0-0000 woled awore ai log Elution profile: G-50 Sephadex 10 15 Volume (ml) 5 10 15 Volume (ml) -A 280 nm --A 410 nm 20 25 A280 --A410 20 Experiment 5. The original protein mixture was loaded onto an SDS-PAGE at pH 8.8. Three protein bands were observed after Coomassie blue staining: 15 000, 30 000 and 45 000. 25

56. 57. 55. A. carboxymethyl cellulose (-ve) at pH 5.0 which proteins would you predict would be eluted If you loaded the original protein mixture (A-E) onto an ion exchange column containing BEFORE the NaCl wash? A. Which protein (A - E) would have the highest ratio of basic residues (lysine & arginine) relative to acidic residues (glutamate & aspartate)? B. C. D. E. B. B. C. C. A. D. D. E. E. Your mixture contains ovalbumin. This protein is colourless with a pl of 4.7 and a molecular weight of 45 000. Which protein (A - E) is ovalbumin? All the proteins None of the proteins Proteins A, B & D Protein C and E Protein A and B m! Protein A Protein B Protein C Protein D Protein E Protein A Protein B Protein C Protein D I snoteid s zi (-A) enxim oy ni amiston or to sno Protein E