Biological Macromolecules: UV‐visible Spectrophotometry

Abstract

Biological macromolecules such as proteins and nucleic acids absorb light in the UV‐visible region of the spectrum. Absorbance measurements are used for measuring concentrations, for the detection of conformational changes and of ligand binding, and for following enzyme reactions.

Keywords: absorbance; proteins; nucleic acids; enzymes; denaturation

Figure 1.

(a) Ultraviolet absorption spectra of the protein ribonuclease T1. The spectrum of the native protein (in 0.1 mol L−1 sodium acetate, pH 5.0) is shown by the continous line, the spectrum of the unfolded protein (in 6.0 mol L−1 guanidinium chloride in the same buffer) is shown by the broken line. Ribonuclease T1 contains nine Tyr residues, which give rise to the maximum at 278 nm, the single Trp residue leads to the shoulders between 280 and 300 nm. The small contributions of the four Phe residues near 260 nm are barely detectable. (b) Difference spectra between the native and the unfolded protein. The major difference at 287 nm arises from the exposure of the Tyr residues in the unfolded protein, the shoulders between 290 and 300 nm originate from exposure of the Trp residue. Spectra of 15 μmol L−1 protein were measured at 25°C in 1‐cm cuvettes.

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Further Reading

Brown SB (ed.) (1980) An Introduction to Spectroscopy for Biochemists. London: Academic Press.

Cantor CR and Schimmel PR (1980) Biophysical Chemistry, part II. San Francisco: W. H. Freeman.

Harris DA and Bashford CL (1987) Spectrophotometry and Spectrofluorimetry: A Practical Approach. Oxford: IRL Press.

Pace CN, Vajdos F, Fee L, Grimsley G and Gray T (1995) How to measure and predict the molar absorption coefficient of a protein? Protein Science 4: 2411–2423.

Schmid FX (1997) Optical spectroscopy to characterize protein conformation and conformational changes. In: Creighton TE (ed.) Protein Structure: A Practical Approach, pp. 261–297. Oxford: IRL Press

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Schmid, Franz‐Xaver(Apr 2001) Biological Macromolecules: UV‐visible Spectrophotometry. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003142]