Question

1) What was the purpose of the following procedures in the current experiment? Your answer will be one or more of the following: purify the protein, determine purity, determine if the protein is properly folded, determine stability, or determine heparin/SOS affinity.

a) Heparin-Sepharose Column Purification:

b) SDS-PAGE:

c) Fluorescence:

d) Circular Dichroism:

e) Trypsin Digestion:

f) Isothermal Titration Calorimetry:

g) Differential Scanning Calorimetry:

h) HSQC NMR:

Answer 1

Protein purification and identification requires a comprehensive utilization of a number of analytical and validatory techniques. These can be identified as below:

a) Heparin-Sepharose Column Purification: This technique is used to extract coarse proein based upon their surface binding properties and can be combined with other column-purification methods to enrich proteins based upon their physico-chemical properties such as surface charge, molecular mass, pI and pH etc.

b) SDS-PAGE: SDS-PAGE is an analytical methods which helps in identification of relative and absolute moleular weights of primary structures of proteins when resolved in a polyacrylamide gel. A molecular weight marker is simultaneously resolved with the test proteins and this helps in analysis of their molecular weights. It can also help in identifying any subunits or polymerization units of the proteins in the gel resolution.

c) Fluorescence: The proteins are made up of amino acids ans some amino acids are enriched with an inherent property of autofluorescence. This property of some amino acids can be exploited to qualitatively analyse the type of amino acids and quantitatively estimate the concentration of these amino acids in the protein by spectrophotometric readings.

d) Circular Dichroism: Circular dichroism is the techniques which helps in analysis of 3-dimensional structure based upon optical properties of the protein and its constituent amino acids. Since amino acids can be levoroatory or dextrorotatory in nature, each amino acid bends the incoming light in a specific manner. This extent and mode of rotation of incoming light can be quantized for each amino acid which can be cumulatively utilized to analyse the nature of protein.

e) Trypsin Digestion:Trypsin digestion takes place at specific polypeptide bonds only and yields amino acid fragments of a native protein. These fragments are often utilized for further segmented analysis so that the whole large protein need not to be assessed at once. The sequences and their properties can be cumulatively sequenced in the end and conjoined to analyse the property of the whole protein.

f) Isothermal Titration Calorimetry: This technique is used to analyse the stability of a protein in a solution. Technically, this physical analytical method can be utlized to analyse the thermodynamic stability of any solute in a solution phase. Since proteins reside in a solution phase in bilogical system, it is very important to know if they are thermodynamically stable in that system. Then only their physico-chemical and biological relavance can be determined. Thus after knowing the structure and 3-Dimensional orientation of a protein, its statbility is determined by isothermal titration calorimetry.

g) Differential Scanning Calorimetry: This method is used to analyse the adiabaticity of a protein in a biological thermodynamic protein. It is very well known that proteins are heat labile by nature. So it remains highly important to know thermal stability and maximum heat which can be provided to and taken from a biological system which can help sustain not only the biological system itself but the proteins might also remain stable. A reference is always used simultaneously to detmine the thermal stability of a protein.

h) HSQC NMR: Heteronuclear Single Quantum Coherence (HSQC) is a high throughput analytical 2-dimensional NMR method which is used to detemine structural properties of a protein at two planes simultaneous with labelled nitrogen (N15) and carbon (C13). This method is more advanced, accurate, precise and realistic when compared to standard NMR analysis which gives only sequence-based information about a protein. The results from this techniques are usually used in structural biology and bio-informatics for analogy to previously known protein domains so that appropriate quarternary protein might be identified.

Thus, a combination of above mentioned techniques is utlized for complete anlaysis of a protein regarding its structural, functional and biological relavance to a system.