Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Quantitative Protein Determination
Spectrophotometric Methods

8.18.8.1. Comparison of absorbance at 280 and 260 nm. Most Proteins exhibit an absorbance maximum at 280 nm due to the presence of Tryptophan and Tyrosine residues. Nucleic Acids, which frequently occur as contaminants in protein solutions, also absorb strongly at 280 nm, but their absorption maximum lies at 260 nm. Warburg and Christian experimentally determined the extinction coefficients of various proteins and nucleic acids at 280 and 260 nm and calculated The ratio of these coefficients (the F factor; Table 8.3); a differential method for protein determination in the concentration range of 50–500 µg/mL has been proposed.

The Warburg and Christian method [388]. The optical density of the protein solution is measured at 280 nm and 260 nm. The ratio of the obtained values is calculated, the corresponding F value is found using Table 8.3, and substituted into the following formula:

Protein concentration (mg/mL) = F∙1/d∙A₂₈₀ [where d is the cuvette path length (in cm)] or [212]:

Protein concentration (mg/mL) = 1.55A₂₆₀ — 0.76A₂₈₀

8.18.8.2. Comparison of absorbance at 230 and 260 nm [194].

Protein concentration (µg/mL) = 183A₂₃₀ — 75.8A₂₆₀

These wavelengths were chosen because they correspond to the minimum and maximum absorbance in the nucleic acid spectrum. Minor deviations in wavelength Setting have virtually no effect on nucleic acid determination, whereas protein absorbance at ~230 nm changes by 1% per 0.1 nm change in wavelength. High concentrations of ammonium sulfate, glycerol, and sucrose, which interfere with the Lowry assay, can be neutralized by appropriate additions in the blank sample.

8.18.8.3. Comparison of absorbance at 235 and 280 nm [399].

Protein concentration (mg/mL) = (A₂₃₅—A₂₈₀)/2.51

Note. Nucleic acids do not contribute to the results because they have equal absorbance at 235 and 280 nm. Based on the Amino Acid Composition data of 208 proteins, the average total absorbance of Trp, Tyr, Phe, His, Met, Cys-Cys, and Cys residues was found to be 0.853, and 0.849 for the 8 studied proteins.

The sensitivity of protein concentration determination at A₂₃₅ is 2.82 times higher than that at A₂₈₀. The overall sensitivity of the method is 45% of that of the Lowry assay.

Another determination method is based on measuring the A₂₃₃/A₂₄₀ absorbance ratio [133].

8.18.8.4. Comparison of absorbance at 215 and 225 nm [383]. Prepare a series of dilutions of the protein solution and measure the absorbance of the samples.

Protein concentration (µg/mL) = (A₂₁₅ — A₂₂₅) ∙ 144

Note. NaCl and (NH₄)₂SO₄ do not interfere with the determination [271]. Certain buffers, such as acetate, citrate, barbital, succinate, and phthalate, absorb strongly at 215 nm, but can be used at low concentrations (0.005 mol/L) provided that a blank correction is applied. A critical evaluation of the method is given in [407]; it is recommended for use with both pure proteins and mixtures within a linear concentration range of 1.5–45 µg/mL.

8.18.8.5. Comparison of absorbance at 280 and 205 nm [335]. The protein solution is diluted with 0.1 M K₂SO₄ containing 5 mM KH₂PO₄, adjusted to pH 7.0 (by adding KOH). The absorbance of this buffer at 205 nm is 0.08 absorbance units/cm and depends on the CO₂ content. Adsorption losses of proteins are minimized by preparing solutions in polyethylene vessels. Quartz cuvettes must be cleaned with concentrated nitric acid, and an appropriate correction for far-UV light scattering should be made.

The major contribution to absorbance at 205 nm comes from the side chains of Trp, Tyr, Phe, and His residues, and to a lesser extent from Arg, Met, and Cys residues. The correction for Trp and Tyr (which is generally omitted for Other Amino Acids) is determined by measuring the absorbance at 280 nm (using a sample with a higher concentration).

Class="center">ε₂₀₅ = 27.0 + 120 ∙ (А₂₈₀/А₂₀₅)

Note. In the spectrophotometric quantification of 12 proteins, the values found were ε₂₈₀ = 0.50–2.65 and ε₂₀₅ = 29.0–36.4. Only in cases of an unusually high Phe content in the sample did the measurement error exceed ±2% [335].

Protein analysis at 205 nm requires a high-grade spectrophotometer, and it should be noted that even trace amounts of compounds absorbing in this region (HCOOH, CH₃COOH, CH₃COCH₃, etc.) must be excluded from the solution.