Section Advances in Biotechnology

PROTEIN AND PEPTIDE DETERMINATION BASED ON THE MODIFIED

BIURET PROCEDURE: IMPLICATIONS FOR VARIOUS
BIOTECHNOLOGIES

PhD Asst. Elena Mihalcea1,

Prof. Dr. Gabi Drochioiu1,
PhD Asst. Stefania-Claudia Jitaru1,
Dr. Violeta Mangalagiu1,2,
Assoc. Prof. Dr. Robert –Vasile Gradinaru1
1
Faculty of Chemistry, Al. I. Cuza University of Iasi, Romania
2
Faculty of Food Engineering, Stefan cel Mare University of Suceava, Romania

ABSTRACT
Spectrophotometric methods for total protein analysis are generally simple, rapid and
sensitive. Such sensitive protein assays may have applications in forensic science, in the
detection of protein contaminants in drugs and in a number of other applications of
research interest. Biuret reaction with proteins and peptides is widely used in clinical
and biological laboratories. In this work, instead of copper sulphate, sodium hydroxide
and Seignette salt, we used insoluble copper phosphate, potassium or sodium hydroxide
and ethyl alcohol for the preparation of the biuret reagent. Absorbance of the biuret
complex was recorded both at 219-230 nm (after dilution) and around 550 nm against a
reagent blank. Amino acid interference was investigated around 550 nm at the same
concentration as proteins. The sensitivity of the method at 226 nm was greater than
those of other spectrophotometric assays (old biuret method, Lowry, and BCA) with a
LOD of about 0.5 µg mL−1 BSA. The new variants of the biuret method for total protein
analysis eliminate the need for precise reagent addition and vortexing inherent in the
widely used Lowry method, providing flexibility of application. The method developed,
which uses an alkaline-alcoholic reagent and insoluble copper phosphate, is simple,
rapid, reproducible and sensitive; it is not influenced by detergents, solvents and buffers
containing ammonium and is flexible enough to change the analytical protocol when
necessary. A discussion was made on the applications of protein and peptide
determination with the new biuret assay.
Keywords: Protein determination, Peptide determination, Biuret reaction, Amino
acid interference, Spectrophotometry

INTRODUCTION
There are numerous methods for protein quantification. Protein determination
assays use progressively smaller amounts of material and modifications to classic
protocols were also introduced to provide a microvolume alternative to traditional
cuvette‐based methods [1-3]. However, classical protein assays are also widely used in
biotechnology and medical laboratories around the world [4,5]. Protein
concentrations in various

113
https://doi.org/10.5593/sgem2022/6.1/s25.14