Assessment for Chromatography / Viral Cuts / Desalting

Question
1. Give at least three reasons on why proteins are considered to be an important
compound for the human body.
2. Name at least five functions of proteins
3. What does exactly happen during protein denaturation?
4. Explain the principles of column chromatography
5. Explain the difference between ion exchange and gel filtration on protein purification

Answer
1. Because proteins play great roles in human body metabolism. At the smallest scale,
even the cell mitosis need a set of protein that ensure the process can occur. Other
proteins important role in the human body is as regulating factor that ensure certain
metabolism can successfully maintain in homeostatic condition. For example of this
metabolism is erythropoiesis. When defect or disruption of this process happened,
blood regulation may be abnormal, production of erythrocytes decrease and can affect
whole body stability. At last, generally why protein is so important is that our body
need it. It used as hormones component, enzyme, building blocks of muscles,
cartilage, bones, and blood. It is used as growth factor and rebuild damage tissues
2. Proteins naturally has wide range of function in life. Protein can be a form of receptor
that present at the surface of the cell membrane. This accommodate cell signaling,
especially in hormonal regulation. Protein can be a transporter agent that brings
oxygen and carbon dioxide through the body in breathing system. Protein also acts as
catalyst or enzymes that help accelerate biological process by decreasing the needed
energy. Protein can also be a form of antibody that helps in immune system. Another
function of the protein is as cell cytoskeleton (microfilament, microtubules,
intermediate filament) that enable body movement.
3. During denaturation, the bonding or interaction that makes secondary, tertiary, and
quarternary structures of protein is disrupted. These including ionic bonds, hydrogen
bonds, disulfide bonds, salt bridges, or hydrophobic interactions. So that, the only
remaining bonds is the polypeptide bonds that makes primary structure. Denaturated
protein lose their biological activity, because of the shape, the active sites that may
correlate to the function is absent. This can happen because of exposure such as heat
or radiation.

4. It is separation technique that utilize column containing small beads as stationary

phase and eluent as mobile phase. Separation of loaded sample happen when some
samples components interacts differently with the stationary phase and while at the
same time the mobile phase bring them through the column at certain flow rate. One

component will be eluted first than the other. At the end, these components will be
analyzed by detector, it may be spectrophotometer or other detector. This part will
detect the absorbance of the eluted components from the sample. Chromatogram is
the main result obtained from the technique. In this result, information such as
retention time and concentration of the samples can be found here.
5. Ion exchange chromatography (IEX) utilize protein net surface charge as the
separation principle, since protein has different net charge. In IEX, the column
consists of charged medium (can be anionic or cationic) as the stationary phase and
buffer as the mobile phase. Interaction between charged molecules and medium are
controlled by the buffer in terms of elution process and separation. While gel filtration
(GF) utilize protein molecular size or weight as the separation principle. In GF,
stationary phase is porous matrix and buffer as the mobile phase. Protein that has
greater size will be eluted first and the smallest size will be eluted last.

Reference:
Anonim. 2004. Cytoskeleton Tutoria: Microtubules, Microfilaments, and Intermediate
Filaments.
http://www.biology.arizona.edu/cell_bio/tutorials/cytoskeleton/page1.html.
Accessed: November, 22nd 2015.
GE Lifescience. 2014. Size Exclusion Chromatography, Principles and Methods. pp. 15-16.
GE Lifescience. 2014. Ion Exchange Chromatography & Chromatofocusing, Principles and
Methods. pp. 11-12.