Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm

Abstract

Quantitative studies in molecular and structural biology generally require accurate and precise determination of protein concentrations, preferably via a method that is both quick and straightforward to perform. The measurement of ultraviolet absorbance at 280 nm has proven especially useful, since the molar absorptivity (extinction coefficient) at 280 nm can be predicted directly from a protein sequence. This method, however, is only applicable to proteins that contain tryptophan or tyrosine residues. Absorbance at 205 nm, among other wavelengths, has been used as an alternative, although generally using absorptivity values that have to be uniquely calibrated for each protein, or otherwise only roughly estimated. Here, we propose and validate a method for predicting the molar absorptivity of a protein or peptide at 205 nm directly from its amino acid sequence, allowing one to accurately determine the concentrations of proteins that do not contain tyrosine or tryptophan residues. This method is simple to implement, requires no calibration, and should be suitable for a wide range of proteins and peptides.

Figure 1

Testing the stoichiometry of 2Ca²⁺-CaM_1–76 binding to the skMLCK M13 peptide. Fluorescence anisotropy was measured for the tryptophan of skMLCK alone (10 µM) and in the presence of 0–50 µM CaM_1–76. Experimental data (average of three measurements) are plotted as filled-in squares, with error bars indicating one standard deviation. The best-fit line is shown in red. The CaM_1–76:skMLCK stoichiometry from the fit of the data is n = 2.13 ± 0.17, which is in excellent agreement with the literature value of 2:1.¹⁴ The titration was performed with [skMLCK] >> K_D to most accurately determine the stoichiometry. The effective K_D from the fit is 100 ± 28 nM, several orders of magnitude weaker than the value determined for full-length CaM (∼50 pM).²⁰