Question

Biochemistry 3. How to analyze the concentration and molecular weight of proteins?

Answer 1

Proteins are polymers . They are polymers of amino acids. There are twenty different types of amino acids occur naturally in proteins.

The Protein concentration can be analysed by following methods:

Kjeldahl method
Food is digested with a strong acid so that it releases nitrogen. The amount of protein present is then calculated from the nitrogen concentration of the food.Thus methosd is used as satandard method of determining protein concentration. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein). The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration.
UV-visible spectroscopy
This method use the natural ability of proteins to absorb light in the UV-visible region of the electromagnetic spectrum or they chemically or physically modify proteins to make them absorb (or scatter) light in this region. The basic principle behind each of these tests is similar. First of all a calibration curve of absorbance (or turbidity) versus protein concentration is prepared using a series of protein solutions of known concentration. The absorbance (or turbidity) of the solution being analyzed is then measured at the same wavelength, and its protein concentration determined from the calibration curve. The main difference between the tests are the chemical groups which are responsible for the absorption or scattering of radiation, e.g., peptide bonds, aromatic side-groups, basic groups and aggregated proteins.

IR SPECTROSCOPY
Infrared techniques can be used to determine the concentration of proteins in food samples. Proteins absorb IR naturally due to characteristic vibrations (stretching and bending) of certain chemical groups along the polypeptide backbone. Measurements of the absorbance of radiation at certain wavelengths can thus be used to quantify the concentration of protein .

NMR SPECTROSCOPY
NMR spectroscopy can be used to determine the total protein concentration of foods. The protein content is determined by measuring the area under a peak in an NMR chemical shift spectra that corresponds to the protein fraction.

The Molecular weight of proteins can be analysed by size exclusion chromatography.

Size-exclusion chromatography

It is a chromatographic method in which molecules in solution are separated by their size. It is usually applied to large molecules or macromolecular complexes such as proteins.

In this protein solution is poured into a column which is packed with porous beads made of a cross-linked polymeric material . Molecules larger than the pores in the beads are excluded and move quickly through the column whereas the movement of molecules which enter the pores is retarded. Thus molecules are eluted off the column in order of decreasing size. Beads of different average pore size are available for separating proteins of different molecular weights. Molecular weights of unknown proteins can be determined by comparing their elution volumes Vo, with those determined using proteins of known molecular weight: a plot of elution volume versus log(molecular weight) should give a straight line.