An improved automated biuret method for the determination of microgram protein concentrations

✍ Scribed by Kenneth V. Honn; Walter Chavin

• Publisher: Elsevier Science
• Year: 1975
• Tongue: English
• Weight: 229 KB
• Volume: 68
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(75)90699-5

✦ Synopsis

The inherently simple biuret reaction is hampered by lack of sensitivity and reproducibility in the determination of low protein concentrations. A method utilizing a modified biuret formulation and colorimeter range expander permits protein determination in the 0.01-3.0 mg/ml range at a sampling rate of 60/hr without significant sample interaction. This inexpensive method is useful in investigations involving large numbers of samples with microgram protein content.

Alterations of many tissue parameters (i.e., enzyme activity, cyclic nucleotide content, steroid output) are conveniently expressed in terms of protein content. The advent of automated techniques permits assays to be performed in large numbers with small sample size. Such approaches necessitate automated protein analysis in the microgram to milligram range. The biuret reaction is conveniently suited to rapid protein determination because of the reagent simplicity, low cost, rapid color development, and long shelf life. However, existing microbiuret procedures have not been automated and the available reagent (1) does not have the storage qualities of other formulations (2, 3). Therefore, alternate procedures (4, 5) have been developed for microgram protein determinations utilizing the more sensitive Folin-Ciocalteau reagent. Automation of a sensitive biuret determination is handicapped by a slow sample rate (30/hr) and insensitivity (2-20 mg/ml) in the microgram range (6). In the present study, an automated biuret procedure was developed using a modified reagent formulation and range expander thereby permitting protein determinations in the 0.01-3.0 mg/ml range.

Methods

Protein concentrations were determined automatically with a Technicon Autoanalyzer. The flow diagram for automated protein assay (Fig. 1) was modified from the Technicon N-14b manifold (7). Measurements 1 Contribution number 339, Department of Biology.