Simultaneous Determination of Protein and Essential Amino Acids in Biological Samples

The utilization of spectrophotometric methods for the determination and analysis of proteins is of great interest as they are generally simple, rapid and with high sensitivity. These protein assays have many uses in the food, plant breeding, pharmaceutical and biomedical industries, as well as in forensic science and a variety of other research fields. Among these, the biuret method is highly sensitive and is widely used in clinical and biological laboratories. However, the use of copper sulphate in the traditional biuret method of protein identification can lead to substantial interference from copper ions. In this framework, we modified the traditional biuret method by using an alkaline-alcohol reagent and insoluble copper phosphate; the protein-copper complex could also be evaluated at 545 nm. In addition, aliquots of the same solution are treated with specific reagents to determine two essential amino acids; tryptophan reacts with glyoxylic acid and lysine is treated with a ninhydrin-based reagent. Calibration curves must also be made at three wavelengths, with absorbance values in relation to the concentration of each component measured by standard methods. The advantage of this new approach is that it requires only 50 mg of ground seed meal to quantify three different components in each sample.