Introduction Pharmaceutics is the science of preparing and dispensing drugs. Drugs, specially vaccines and antibiotics contain proteins in the form of enzymes or antibodies as the active component. In most cases these foreign proteins can be dangerous or harmful in larger concentrations for the body. Therefore it is crucial for the accurate and precise estimation of the protein concentration in the final product. This estimation of proteins is not only important for the final product but it also helps in the optimization of the production. The quantification of proteins at each step of a new procedure helps to identify
• the degree of purification in each step
• the functional step that is critically dependant on the protein concentration…show more content… Estimation by UV-VIS spectrometry
1.1. ESTIMATION OF PROTEIN BY BIURET METHOD
PRINCIPLE:
Biuret assay is one of the basic assays used for protein concentration estimation. The biuret reagent consists of sodium hydroxide, hydrated copper (II) sulphate, together with potassium sodium tartrate. The tartrate is used to stabilize the cupric ion by complex formation.
During the reaction, the cupric ions react with the nitrogen atoms in the peptide bond in an alkaline solution to form a violet coloured complex, with absorption maxima at 565nm wavelength.
The intensity of the colour formed is proportional to the amount of protein in the sample. Thus using Beer – Lambert law, the concentration of protein is estimated by measuring the absorbance of standard concentrations, plotting the standard curve using a least square error fit and plotting the unknown concentration on the curve. The reagent is added to the sample and incubated for 30min in room temperature. The absorbance is measured at 450nm, and the concentration is determined by the use of beer-lambert law. This reaction is the first step in other spectrometric assays like Lowry’s, BCA, etc.
Materials…show more content… ESTIMATION OF PROTEIN BY LOWRY’S METHOD
PRINCIPLE:
Similar to biuret assay, Lowry’s assay is one of the most cited procedures for the estimation of proteins. In addition to the copper (II) complex, the Lowry’s reagent contains a mixture of phosphomolybdate and phosphotungstate called Folin’s reagent.
During the reaction, the cupric ions react with the nitrogen atoms in the peptide bond in an alkaline solution to form a violet - blue coloured complex. The blue colour is further intensified by the reaction of the Folin’s reagent with the phenolic groups. The final product has an absorption maximum around 640nm. The intensity of the colour formed is proportional to the amount of protein in the sample. Thus using Beer – Lambert law, the concentration of protein is estimated by measuring the absorbance of standard concentrations, plotting the standard curve using a least square error fit and plotting the unknown concentration on the curve.
Materials Required Sodium hydroxide, sodium carbonate, hydrated copper sulphate, potassium sodium tartrate, Folin’s reagent, known protein standards or stock solution, UV/VIS Spectrophotometer, pipettes, cuvette.
Preparation of Lowry’s reagent Lowry’s reagent has 4 parts: A, B, C,