Question

1. Joshua used gel filtration chromatography to purify protein Z from crude homogenate prepared from Arabidopsis plants. He noticed that protein Z appeared to have eluted from the column in two separate peaks. Using a standard curve, he calculated the MW of protein Z eluted in the first peak to be 156 KDa, and the protein Z eluted in the second peak had the apparent MW of 78 KDa. Which one do you think is the correct MW? What does this tell you about the structure of the protein?

2. Read the following paragraph and correct all errors (4.15 points).

An unemployed high school student, Summer, has isolated a protein from a juniper tree with an overall negative charge. He performs ion exchange chromatography using a cationic resin with blue dextran to aid the proteins association with the column. As the proteins come off the column, the spec shows 4 peaks, only the first peak (wash peak) shows activity in an enzyme assay. Since all fractions associated with this peak had activity, they were pooled and total protein assayed. Total protein analysis was assayed using Bicine reagent which interacts with proteins to cause a change in absorption when measured with a spectrophotometer. The results of the total protein calculation showed that there was a modest decrease in total protein which means that there was a large amount of pure protein of his interest present in the sample.

3.You have accidentally mixed a bunch of amino acids (tryptophan, tyrosine, alanine, selenocysteine, proline, arginine) together and once your boss finds out, you will be fired on the spot. Your only hope of saving your job is to separate them before anyone finds out. You decide that the only viable option is to use ion exchange chromatography. Explain, in detail, how you would do this? (4.15 points)

4. Why should you not lose any LDH in your 20k x g spin? How does this explain an increase the fold purification of LDH in your 20k x g sample compared to your crude homogenate? (2 points)

Answer 1

Ans 2 Corrected Version

An high school student,named summer, had isolated a protein from a juniper tree with an overall negative charge. He performs ion exchange chromatography using a cationic resin with blue dextran to aid the proteins interaction with the column. As the proteins elutes out of the column, the chromatogram depicts 4 peaks, only the first peak (which is the wash peak) shows activity in an enzyme assay. Since all fractions associated with this peak had activity, they were pooled and total protein were analysed. Total protein analysis was carried out using Bicine reagent which interacts with proteins to cause a change in absorption when measured with a spectrophotometer. The results of the total protein calculation showed that there was minimal decrease in total protein which means that there was a high amount of pure protein of his interest present in the sample.

ANS3.

The most simple and effective method to separate out mixture of amino acids is using an Amino acid analyser. Ion-exchange chromatography with post column ninhydrin detection is one of the most common methods employed for quantitative amino acid analysis.

Where Separation of the amino acids on an ion-exchange column is accomplished through a combination of Buffers having different pH and cation strength.

A temperature gradient is often employed to enhance separation.

When the amino acid reacts with ninhydrin, the reactant has characteristic purple or yellow color.

Amino acids, except imino acid, give a purple color, and show the maximum absorption at 570 nm.

The imino acids such as proline give a yellow color, and show the maximum absorption at 440 nm

• The postcolumn reaction between ninhydrin and amino acid eluted from column is monitored at 440 and 570 nm and the chromatogram obtained is used for the determination of amino acid composition.

Alanine being neutral and arginine being positively charged will be eluted first followed by selenocysteine which is sulphur containing amino acid, proline being heterocyclic.

Aromatic amino acids like tryptophan, tyrosine will eluted last based on the interaction with the positively charged column and variations in the buffer with different pH.

ANS 4.

Lactate Dehydrogenase (LDH) is an important enzyme in humans. It occurs in different regions of the body, each region having a unique conformation of different subunits. LDH is a key enzyme in anaerobic respiration. Anaerobic Respiration is the conversion of pyruvate into lactate acid in the absence oxygen.

Human LDH is a quaternary protein formed of the combination of two subunits, M and H (Muscle and Heart) into a structure of four of the subunits. The various combinations found in the human body are:

• (4H) Heart
• (3H1M) Reticuloendothelial
• (2H2M) Lungs
• (1H3M) Kidneys
• (4M) Muscle and Liver

The secondary structure of LDH as shown here is comprised of 40% alpha helices and 23% beta sheets.The SCOP data classifies this form of lactate dehydrogenase as mixed beta-alpha-beta, with mainly parallel beta sheets.

Using the example of lactate dehydrogenase, we show that enzyme quaternary structure has an important influence on the structure of the active site and that models that comprise all amino acids in the vicinity of an active site, but are missing this structural information, can lead to incorrect results. We also show that binding isotope effects are very sensitive to the geometric parameters, and thus one should be very cautious when interpreting results obtained with models that are too coarse. In terms of the type of hydrogen bonds, our results indicate that binding isotope effects are pronounced only when a hydrogen bond exhibits some covalent character.Which explains the Fold purification of LDH when compared to the crude homogenate.