2.521.510.50=Mike has determined that enzyme he is attempting to purify has an isoelectric point of 4.5 (pl:4.5). He has decided to examine anion exchange chromatography as a potential purification step.He tested out using 2 different buffer and linear NaCl gradient on HPLC (as what you did in lab).His results are shown below.pH 8 data1 3 4 5 7 9 10111315171920212325272930Fraction number-A280Enzyme activity2.521.510.50pH 5 data1 3 4 5 7 9 10111315171920212325272930Fraction number-A280--Enzyme activityWhich buffer should Mike uses for his purification (both buffer has pH higher than enzyme's pl)? Why

The isoelectric point (pI) of a protein is the pH at which the net charge of the protein is zero. As the pH goes greater than the pI, the protein becomes more and more negatively charged. Similarly when the pH goes lower than the pI, the protein becomes more and more positively charged. In anion exchange chromatography, negatively charged species (anions) in the mixture gets attached to the positively charged chromatographic column. The anions attached to the column can be later eluted using salts (as in our question where NaCl is used for elution).From the given plot, pH 8 is more suitable for purification due to the following reasons. 1. Specific activity is a measure of the purity of an enzyme in the sample we eluted out. Higher the Specific activity of the eluted sample, greater is the purity. Specific activity = Enzyme activityAmount of protein Here the Absorbance at 280 nm is the relative quantification of the protein i.e. its gives the amount of protein (because proteins absorbs well at 280nm ). To get a high Specific activity value, we need to have a high enzyme activity value and a low amount of protein (look at the equation above). From the data, if you roughly calculate the specific activity, the protein purified from pH 8 buffer has higher specific activity compared to pH 5. Hence buffer with pH 8 is preferred. In simple words, from the data you can see that at pH 8, fractions 24 to 30 have high enzyme activity (enzyme activity curve is high) and low amount of protein (A280 curve is low ). Which means, these fractions have high specific activity i.e. it contains the majority of your protein of interest (highly pure fractions). 2. The protein can be eluted within a frame of 7 fractions (24-30) using pH 8, which is less when compared to 12 fractions (1-12) under pH 5. 3. Also, more of the protein will be negatively charged at pH 8 compared to pH 5. So, more of the protein will get attached to the column at pH 8. Also, the protein will be more negatively charged at pH 8, so it will be attached more tightly with the column. This will reduce the loss of proteins during the HPLC process.