Molecular Biology: A Practical Guide - Velykov V.A. 2013

Spectrophotometry of DNA, RNA, and Protein Preparations
Determination of Protein Concentration via Intrinsic Fluorescence

Spectrophotometric Methods for protein determination are based on measuring the absorption or emission of light in the ultraviolet region of the spectrum. While UV absorption spectrophotometry presents certain challenges when applied to Proteins, it is far more common to determine proteins bound to specific Dyes colorimetrically in the visible spectrum (see Topic 10), or to measure emission in UV light.

Protein solutions exhibit absorption within the wavelength ranges of 270–290 nm and 200–225 nm. Absorption at 280 nm is determined by the presence of aromatic Amino Acids—Tyrosine, Tryptophan, and, to a lesser extent, phenylalanine—in the protein molecule. Absorption at λ=210 nm, which is due to peptide bonds in proteins, is nearly 20 times higher than that at 280 nm.

Since ultraviolet absorption at 210 nm is mainly due to peptide bonds, the absorbance values vary only slightly among different proteins. The determination of total serum protein via direct photometry at a wavelength of 210 nm yields results comparable to those obtained by the biuret and Kjeldahl methods. However, this method is used relatively infrequently due to The Need for UV-transparent cuvettes and a monochromator, which increases the cost of the Procedure.

Compared to photometry at λ=210 nm, the accuracy and Specificity of protein Determination Methods based on absorption at λ=280 nm are rather low, as the content of tyrosine and tryptophan can vary across different proteins. Furthermore, the presence in Blood serum of free amino acids (tryptophan and tyrosine) as well as uric acid and bilirubin—all of which absorb at 280 nm—introduces a certain degree of error. Consequently, this method is not employed for the direct determination of total protein in blood serum. Nonetheless, it is convenient for use with protein preparations of varying degrees of purity (Appendix 17). The presence of Nucleic Acids and NUCLEOTIDES as impurities will interfere with the assay.

In addition to UV Light absorption, emission measurements are also utilized. The method outlined below is useful for very low concentrations of proteins available in limited amounts. Most importantly, it does not require preliminary staining of the resulting protein sample (which is irreversible, see Topic 10), as the protein solution possesses intrinsic fluorescence.

Materials and Equipment

Spectrofluorometer, 3-mL quartz cuvette, micropipette, buffer solution, protein solution in buffer, and a 9-mg sample of bovine serum albumin (BSA) for calibration.

Procedure

1. Obtain the fluorescence spectrum of the test protein solution using an excitation wavelength of 280 nm, with emission scanning in the 310–360 nm range. The scanning parameters (fluorescence mode, read mode, full correction, 1-nm step) are determined based on the fluorescence maximum value with correction turned off (10–90 units). If a peak of fluorescence intensity is observed in the 330–340 nm region (with full correction applied), the concentration can be determined; otherwise, the protein concentration is too low and the sample must be concentrated.

2. Record the fluorescence intensity value I and the emission wavelength X at the maximum of the fluorescence curve. Record the spectrum for the buffer alone (in the absence of protein) using the same parameters. Record the fluorescence intensity value 1₀ at the established wavelength X. Calculate the value I-I_o.

3. Using the buffer solution, prepare a series of serial dilutions of the calibration protein solution with concentrations ranging from 3x100 mg/mL to 3х10-⁶ mg/mL in 10-fold (order of magnitude) steps, each with a volume of 2.7 mL. Determine I-I_o for each solution, and then use MS Excel to construct a calibration curve plotting protein concentration against I-I_o.

4. Determine the concentration of the test protein solution using the calibration curve.