ANALYTICAL BIOCHEMISTRY 68, 230-235 (1975)

An Improved Automated Biuret Method for the Determination of Microgram Protein Concentrations K E N N E T H V . H O N N AND W A L T E R CHAVIN

Department of Biology, 1 Wayne State University, Detroit, Michigan 48202 Received March 3, 1975; accepted April 18, 1975 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. MATERIALS AND 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. 230 Copyright© 1975 by AcademicPress, Inc. All rightsof reproductionin any formreserved.

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Fro. 2, Circuit diagram of range expander, The unit is housed in an aluminum diecast box. A Canon (WK-5-21C 3/8) plug (A) fits into the output socket of the Technicon Model 1 colorimeter. A Canon (WK-5-32S) socket (B) which accepts the autoanalyzer recorder input lead plugs, is mounted on the housing. The potentiometer (C), a Helipot PU5K f~ --+ 2% (Beckman Instr. SC665) is mounted in series with (D) a fixed resistor (Sage Silicohm, SR5W 2.5K ft ~+ 3%). Clockwise rotation of the potentiometer control knob increases the resistance expanding the recorder output.

232

HONN AND CHAVIN

were conducted at 25°C, 550 nm in a Technicon Model 1 colorimeter fitted with a 15 mm tubular flow cell. Amplification of the recorder input was accomplished by inserting a variable resistance (range expander) between the colorimeter and recorder (Fig. 2). This unit was modified from that described by Annan and Fisher (8). All measurements were performed at the potentiometer setting of 1.0. The biuret reagent was modified from Weichselbaum (2) and prepared and stored as two separate stock reagents (A and B) which were mixed immediately prior to use. Stock solution A consisted of cupric sulfate ( C u S O 4 " 5 H 2 0 ) , 30 g/liter; sodium potassium tartrate (KNaC4H406"4H20), 90 g/liter; potassium iodide (KI), 10 g/liter, and sodium hydroxide (NaOH), 1.6 g/liter. Stock solution B consisted of potassium iodide (KI), 5 g/liter and sodium hydroxide (NaOH), 8 g/liter. The working solution consisted of 1 part stock solution A to 4 parts stock solution B. The working solution was filtered (Whatman No. 1 filter paper) and 5 drops 20% Triton X-100 per liter added prior to use. This solution was stable at room temperature for 6-8 hr without detectable change in replicate samples. The wash contained 5 drops 20% Triton X-100 per liter distilled water. Protein standards were prepared from fraction V bovine serum albumin (Technicon, lot no. T 1A 137; Kjeldahl nitrogen, 157 mg N/g) dissolved in 1 N NaOH. Protein standards ranging from 0.01 to 3.0 mg/ml were stored at 4°C.

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An improved automated Biuret method for the determination of microgram protein concentrations.

ANALYTICAL BIOCHEMISTRY 68, 230-235 (1975) An Improved Automated Biuret Method for the Determination of Microgram Protein Concentrations K E N N E T...
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