Rapid biuret protein determination

Aim To become familiar with the biuret method for protein determination To use the biuret method to determine the concentration of protein in human serum

Introduction Albumin is the most abundant protein in the human blood and it is produced by liver. Albumin maintains the fluid of the blood within the vessels, transports vitamins and minerals to the whole part of the body and carries drugs and other substances out of the blood stream. The range value of albumin in the human serum as between 52.2 63.8 and the value out of this range refer to Kidney and liver diseases. Amino acid is the building blocks of albumin and contains amino group (NH3+) and carboxyl group (COO _). Amino acids are linked together by peptide bonds and the longest chains of amino acids are called polypeptide. Biuret reagent is the most common method used to determine the concentration of albumin in the human serum. It consists of copper (II) sulphate and potassium sodium tartrate mixed together in a solution of sodium hydroxide. Biuret is a strongly alkaline; it contains sodium and potassium tartrate which belongs to alkali group.The copper sulfate in the alkaline solution reacts with the polypeptide chains and change color of alkaline solution from blue to purple, the reaction known as a biuret reaction. In the biuret reaction, cupric ions (Cu 2+) react with the peptide bonds and reduction of the copper from cupric ions (Cu2+) to cuprous ions (Cu1+). The first reaction occurs at room temperature and the second reaction occurs during the incubation in water bath for 20 minutes. No change in color occurs when a biuret solution is added to water, but formation of purple color occurs when a biuret is added to the protein. Single amino acid and dipeptide do not react in the biuret reaction, but polypeptide reacts and forms a purple color. The cupric ion (Cu 2+) reacts with 4 to 6 peptides bonds and the more color produced the more peptide bonds participate in the reaction.

When a white light travels through different materials some solutions absorb light. The purple color that formed in the biuret reaction is the unabsorbed light. Biuret reaction has absorbed other than purple color and the human eye can only detect the unabsorbed light. Spectrophotometer is a lab machine is used to measure the amount of the absorbed light at a certain wavelength, it works when the light from the light source pass through cuvettes (special containers used to not obstruct the light passing through it) some of the light is absorbed by the solution; the rest is transmitted through the sample to the detector. The relationship between absorbance to concentration is linear and standard curve is used to measure the unknown concentrations.