Protein Quantitation using a UV-Visible Spectrophotometer – Biuret Method –

December 9, 2020

Introduction

Generally, protein quantitation can be made using a simple UV-Visible spectrophotometer. The V-730 Bio (Figure 1) is a UV-Visible spectrophotometer designed for biochemical analysis. The V-730 Bio includes 6 quantitative methods based on UV absorption spectrophotometry including the Lowry, Biuret, BCA, Bradford, and WST methods.

Figure 1. UV-Vis spectrophotometer (V-730BIO, left) and Quantitative Analysis program (right)

Table 1 (below) shows the features of the six different quantitation methods. As outlined, a method can be selected by reviewing the sample and quantitation range and the presence of possible contaminants. Five of the analysis methods use a chromogenic reaction. Reagent manufacturers produce chromogenic kits for BCA, Bradford or WST with a protocol explaining the relevant measurement procedures *1). Chromogenic reagents for the Lowry and Biuret methods must be prepared by the user. The measurement procedures for the Lowry and Biuret methods differ according to use. For that reason, this application note explains how to create the calibration curves for the Lowry, Biuret and UV absorption analysis methods. Fluctuations in the values depending on the type of proteins and the differences in the concentration ranges calculated as a result of the cell volume *2) were also investigated.

*1) BCA method: BCA Protein Assy Kit – by PIERCE
Bradford method: Protein Quantification Kit – Rapid by DOJINDO
WST method: Protein Quantification Kit – Wide Range by DOJINDO

*2) 10 mm rectangular cells (quartz) and disposable cells (Figure 2) were used for the experiments. The optional cell holder accessory enables the measurement of a sample with a small volume (minimum volume: 100µL)

Figure 2. Disposable cuvette

Table 1. Details of the Protein Quantitation Methods

Principle Concentration
Range
Advantages Disadvantages
UV Abs The absorption maximum at 280 nm corresponds to the response of the tyrosine and tryptophan and is used for the analysis method. 50 to 2000µg/mL (*BSA) Simple method. Sample can be used after measurement. The absorbance differs for each protein. Proteins such as collagen and gelatin that do not have absorption at 280 nm cannot be measured. Contamination by nucleic acids with absorption in the UV region obscures the measurement.
Biuret Protein solutions turn purple after the polypeptide chain chelates with a copper ion. An alkaline solution of Biuret reagent including copper sulfate and Rochelle salt is added to a protein solution. Uses the absorption maximum at 540 nm to determine the quantity. 150 to 9000µg/mL (BSA) Simple procedure. The chromogenic rate is constant for most proteins. Low sensitivity. Sample with a low protein concentration cannot be measured. The chromogenic reaction is influenced by high-concentrations of trisaminomethane, amino acids, and ammonium ion.
Lowry Add an alkaline copper solution to a protein solution. Tyrosine, tryptophan, and cisteine of proteins reduce molybdenum acid and phosphotungstic acid of a phenol reagent, turning the solution blue. Uses the absorption maximum at 750 nm to determine the quantity. 5 to 200ug/µL (BSA) High sensitivity. Widely used. Complicated procedure with a long preparation. Since the chromogenic reaction occurs by a reduction reaction, contamination of the reduction material interferes with the quantitative determination. The chromogenic rate differs for each protein.
BCA The BCA method combines the Biuret method and Bicinchoninic Acid (BCA). BCA has high sensitivity and selectivity for copper ions. When a copper ion that is formed by the reduction action of protein reacts with 2 molecules of the BCA, the solution turns purple. Uses the absorption maximum at 560 nm to determine the quantity. 20 to 2000ug/µL (BSA) Simple procedure. High sensitivity with wide concentration range. Thiol, phospholipid, and ammonium sulfate interferes with the measurement.
Bradford The absorption maximum of a protein shifts from 465 nm to 600 nm when the protein binds to the Coomassie Brilliant Blue G250, one of triphenylmethanse blue pigment. Uses the absorption maximum at 600 nm to determine the quantity. 10 to 2000g/µL (BSA) Very simple operation. Hardly influenced by the blocking materials. The chromogenic rate differs for each protein. Contamination by a surfactant can interfere with the chromogenic reaction.
WST Reduce WST-8 with proteins with a high pH, turning the sample blue. Uses the absorption maximum at 650 nm to determine the quantity. 50 to 5000µg/mL (BSA) Simple method. Hardly affected by a surfactant. The chromogenic rate differs for each protein.

*BSA: Bovine Serum Albumin

Biuret Method

Protein solutions turn purple with an absorption maximum of 540 nm when Biuret reagent is added (Figure 3).

Figure 3. Color change after adding Biuret regent

The time course of the chromogenic reaction was measured at 540 nm absorption. The absorbance became stable approximately after 60 minutes (Figure 4). Figure 5 shows the spectra of the HSA aqueous solutions that were measured after 60 minutes. The Biuret method determines the quantity by using the absorption maximum at 540 nm after reacting with the sample for 60 minutes.

Figure 4. Time course of the chromogenic reaction

Figure 5. Spectra of the HSA solutions using the Biuret method

Chromogenic Reagent
Biuret reagent: Add 60 mL of 10% NaOH to 100 mL of aqueous solution to dissolve 0.3 g CuSO4 and 1.2 g Rochelle salt, and then add water to make up to a total volume of 200 mL. (The standard reagent can be stored in a polyethylene bottle.)

Samples

Bovine Serum Albumin (BSA): 0, 0.25, 0.5, 1, 5, 9 mg/mL (10 mm rectangular cell (quartz))
0, 1, 3, 5, 9 mg/mL (Micro cell)
Hen Egg Lysozyme: 0, 1, 3, 5, 9 mg/mL (10 mm rectangular cell (quartz), Micro cell)
Chymotrypsin from bovine pancreas: 0, 1, 3, 5, 9 mg/mL (10 mm rectangular cell (quartz), Micro cell)

Measurement Procedures
Add Biuret reagent (2.0 mg) to 500 µL of protein aqueous solution and mix them well. Then react the solution for 60 minutes. Measure the absorbance at the wavelength of 540 nm.

Table 2. Results for the Biuret Method

Protein Cell Concentration Range Calibration Curve Formula Correlation Function Standard Error Detection Limit Determination Limit
BSA 10 mm rectangular cell (quartz) to 9 mg/mL Y = AX2 + BX + C
A = -0.0005 ± 4.3444 x 10-5
B = 0.0548 ± 0.0004
C = 0.0505 ± 0.0004
1 0.0109 0.008 mg/mL 0.1483 mg/mL
Micro cell to 9 mg/mL Y = AX2 + BX + C
A = -0.0016 ± 0.0003
B = 0.0653 ± 0.0027
C = 0.0450 ± 0.0045
0.9998 0.0775 0.0696 mg/mL 1.0127 mg/mL
Lysozyme 10 mm rectangular cell (quartz) to 0.9 mg/mL Y = AX2 + BX + C
A = -0.0009 ± 4.9343 x 10-5
B = 0.0591 ± 0.0005
C = 0.0509 ± 0.0008
1 0.0144 0.0133 mg/mL 0.2420 mg/mL
Micro cell to 9 mg/mL Y = AX2 + BX + C
A = -0.0053 ± 0.0005
B = 0.0983 ± 0.0048
C = 0.0476 ± 0.0063
0.9999 0.0786 0.0640 mg/mL
α-Chymotrypsin 10 mm rectangular cell (quartz) to 9 mg/mL Y = AX2 + BX + C
A = -0.0012 ± 0.0004
B = 0.0064 ± 0.0035
C = 0.0578 ± 0.0060
0.9996 0.0958 0.0934 mg/mL 1.2987 mg/mL
Micro cell to 9 mg/mL Y = AX2 + BX + C
A = -0.0010 ± 0.0003
B = 0.0614 ± 0.0025
C = 0.0343 ± 0.0043
0.9998 0.0697 0.0695 mg/mL 0.9987 mg/mL

Keywords

Protein concentration, protein quantitation, colorimetric assay, Bradford, BCA, Lowry, Biuret, 260/280

About the Author

Spectroscopy Group