5. The following table shows show results obtained in a purification of lactate

Question

5. The following table shows show results obtained in a purification of lactate dehydrogenase from crocodile muscle. Volume Protein Enzyme (mL) conc. activity Specific Overall (mg/mL) U/mL) activity purification (U) (U/mg) 0% of starting activity) Stepl Crude extract 50 10 400 n/a Step 2 Supernatant after PH 5.6 precipitation 340 Step 3 Pooled active fractions from anion exchange column 25 600 Step 4 Pooled fractions from coenzyme affinity column 6,000 With use of data shown in the table, calculate Gi) the specific activity (U/mg) at each stage, (i) the overall purification achieved at each stage, (ii) the yield at each stage and as % of the starting activity. (2.5 marks) Write your answers in the table under the appropriate columns. a. (3 marks) (3 marks)

Explanation / Answer

a) i) specific activiity= enzyme activity / protein concentration

step 1 specific activity = 400/10 = 40 U/mg

step 2 specific activity = 340/6 = 56.67 U/mg

step 3 specific activity = 600/1 = 600 U/mg

step 4 specific activity = 6000/2 = 3000U/mg

ii) overall purification = specific activity in each stage / initial specific activity

stage 1 purification = N.A

stage 2 purification = 56.67/40= 1.42

stage 3 purification = 600/40 = 15

stage 4 purification= 3000/40=75

iii) yield (U) can be calculate as enzyme activity x volume

stage 1 yield = 400 x 50 =20,000U       % of starting activity = (20,000/20,000) x100 = 100 %

stage 2 yield = 340 x 55 =18,700U        % of starting activity= (18,700/20,000) x100 = 93.5%

stage 3 yield = 600 x 25 =15,000U        % of starting activity= (15,000/20,000) x100 = 75%

Stage 4 yield = 6000 x 2 =12,000U        % of starting activity= (12,000/20,000) x100 = 60%

B) From the table we can infer that in each successive stage specific activity of enzyme is increasing this is due to fact that in each successive stage undesired protein are seperated out which can be infered form the overall purication column that clearly suggest that overall specific activity has increased by 75 fold .

the yield column signifies how much desired protein is being recovered.

C) inhibitor-based studies and/or substrate-based studies are required to further validate enzymatic purity. Combinations of these methods can also be used to enhance confidence in the assay. Enzymatic purity can be highly substrate and format dependent; that is, the same enzyme preparation can be used with one substrate/format and have 100% of the detected activity come from the intended target enzyme, but a different substrate or format may reveal multiple enzyme activities that are present in the preparation.

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