Question

Based on the results in the table, is anion exchange a good purification step, yes or no. Defend your answer. Could someone please explain how you would know if the purification worked looking at a table like this? Which values would change?

Dous lloded during the way to Proteins in eine Wrn that once the non-poc b. A student is purifying an enzyme wid heyme which catályzes the Oxidation of alcohol functional groups Gising NAD as a cofactor. NADH t e desechipekun forms as a product; NADH is a chromophore with a 2 Melute with that at 340 nm and an extinction coefficient of 12.7 mM cm Please complete the following purification table based on the information provided below. Total Protein Volume Concentration Total Protein Bar Total Protein Activity per Specific Sample Reaction ml protein Total Units Activity mg/ml) (mg Rate (Abs/s) (U/ml) Crude Extract 155 1.95 (U) (Ums Antion Exchange 87 0.79 1302.25 68.13 15ml 1,95 my 8 81 me x 99 mg 0.0032 9,071 14060014,6518! 0.110 31.18 2712,76 39.47 | 200) 9 .0080 ) 005 4.071u 155 Assay conditions Total Reaction volume: 3.0 ml Alcohol substrate saturating NAD substrate saturating Pathlength: 1 cm Beer's Law: Abs = cct Assay Volume used: 5.0 c. Based on the results in the table, is the Anion exchange a good purification steps or no. Defend your answer in 1-2 sentences I would say no because the humbers) seem to be too high. Either that means the purification was flowress, or there is contamination giving it High readings, more than I likely its contamination, Points

Answer 1

Purification should yould a sample containing only one type of molecule. Cie Anion exchange Chromatography Purified) The protein sample may be only a praction of 1% of Starting material (recrude) & Enzyme activity - Ability of enzyme to prom particular reaction. In this case, enzyme catalyse alcohol functional groups and generati reduced NADH which absorb light at 340nm. whereas nast does not. Our assay for enzyme activity (U/ml) during purificatia of is this the increase in absorbance of light at 340 obserued in 1 min. - also To analyze the purification is effective, we need amount of protein (ing) By two values ume & mg of protein (Purified) , we con calculate specific activity (mg). → specific activity will rise as the purification proceeds which means protein mixhure contains protein of interest to a greater extent. -> Conclusion :- Overall goal of Purification is to maximize the specific activity (ung).

So, yes Anion exchange is a good purification step because specific acitivity of purified enzyme increases upto nas % (pom 4.6 ung to 39.47 8/mg) (DON'T Consider proteni losses which is only 77%) Another point to consider is your recovery of total protein (mg) is good but your activity ang is low this mene exhibit it poor purification. As mentioned earlier purification quality depends ou specific activity (uling) of enzyme (Parified protei).