Question

Background:

2. Phenylketonuria (PKU) is an inherited disease which results from the lack of the enzyme phenylalanine hydroxylase (PAH). The PAH enzyme catalyzes the first step in the degradation of Phe. In PKU patients, Phe accumulates and is eventually transaminated to phenylpyruvate. Excess phenylpyruvate accumulates in the blood and urine and has the effect of causing mental retardation if untreated. Screening programs identify PKU babies at birth, and treatment consists of a low Phe diet until maturation of the brain is completed. It is known that the PAH enzyme contains 451 residues and has a MW of 51.9 kDa. More than 60 different mutant genes giving rise to nonfunctional PAH proteins have been identified in PKU patients.

Question

1. In order to learn more about the PAH enzyme, it was necessary to purify it. PAH has been isolated
from both rats and humans. In the rat, three isozymes of PAH have been identified in the liver. Their
molecular weights are identical, but their charges are different, as demonstrated by isoelectric
focusing. The pI values are 5.2, 5.3 and 5.6. DEAE-cellulose (anion exchange) chromatography was
one of the steps in the purification procedure of the enzymes. Predict the order of elution of these
isozymes from the DEAE-cellulose column. What pH buffer would you choose in running the
column?

( please type the answer to make it clear )

Answer 1

I believe the correct order of elution would be

1) 5.6

2) 5.3

3) 5.2

you can use any buffer that has a pH more than that of 5.6 so that at that pH, all the proteins would be negatively charged and attach to the positively charged column. decreasing the pH would bring about pI of the proteins at which they will dissociate from the column in a sequential manner.

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