Take-home pre-lab questions for 2-16 AND 2-21 (4 points total) The following dat

Question

Take-home pre-lab questions for 2-16 AND 2-21 (4 points total) The following data describe two very similar dehydrogenase enzymes found in the cytosol of rat liver cells. We used four techniques so far in exercise 4 to purify the rat lactate dehydrogenase (LDH) enzyme hydrophobic affinity chromatography, anion exchange chromatography, size exclusion chromatography, and an enzyme activity assay. If you wanted to purify rat alcohol dehydrogenase instead of LDH, how would you change each of the four techniques to target the alcohol dehydrogenase enzyme? If you don't think you need to change anything then describe what results you would expect and how they would be different from what you got with LDH. (1 point for each technique) Rat Alcohol dehydrogenase 1 (ADH1) pl: 8.52 Molecular weight (average): 39,514 Da Reaction catalyzed: ethanol NAD >acetaldehyde+ NADH Cofactors: NAD' and Zn Rat Lactate dehydrogenase (LDH): pl: 6.8 Molecular weight (average): 36,612 Da Reaction catalyzed: pyruvate + NADHlactate NAD Cofactors: NADH 1. Hydrophobic affinity (aka Cibacron Blue) chromatography 2. Anion exchange (aka DEAE) chromatography 3. Size exclusion (aka gel filtration) chromatography 4. Enzyme activity assay

Explanation / Answer

In this question the both the enzyme are dehydrogenase hence they might be share the structurally same domain, hence, cibaron blue can be used in this molecular weight is same. So in this case same hydrophobic material can be used for the purification of LDH.

In anion exchange chromatography pI of ADH is 8.6 and whereas LDH is 6.8 pI. In this situation buffer needs to change. As you know that at below pI protein contain positive charge and above pI protein contain negative charge. Hence, we need a buffer that provide the negative charge to our protein and should be 1 digit above the pI. Positively chage, that is same like DEAE, will use such as tris, piperazine etc which having pH 7.8 can be used for the binding and purification of LDH.

Here the size of LDH and ADH is same hence same gel filtration material can be used but buffer that is used for purification is should be below or above the pI of protein.

Enzyme activity will be used for the specific activity or for purity determination. But here reaction is just opposite to the ADH where NADH is oxidizing to NAD. As we know that NADH will absorb light at 340 nm hence decrease in absorbance at 340 nm will use for the determining the enzyme activity.

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