4.6. Spectrophotometry in practice: some examples

4.6.1. Absorption spectrum of ATP

The absorption maximum of the molecule is at 260 nm due to its aromatic structure (Figure 4.5). As mentioned above, this peak at 260 nm is also present in the absorption spectra of nucleic acids.

Absorption spectrum of ATP

Figure 4.5. Absorption spectrum of ATP

4.6.2. Hyperchromicity of DNA

The absorption maximum of nucleic acids at 260 nm originates from their constituent aromatic groups. High temperature causes DNA to “melt”—hydrogen bonds connecting the bases start to break and absorbance at 260 nm rises. This phenomenon is known as hyperchromicity (Figure 4.6). The transition temperature is also called “melting temperature” (Tm).

Hyperchromicity of DNA

Figure 4.6. Hyperchromicity of DNA. A, Absorption spectra of a DNA molecule at 30°C (red) and 90°C (black). B, Temperature-induced change in absorbance at 260 nm, reflecting the denaturation (melting) of the double-stranded DNA structure.

4.6.3. Absorption spectra and molecular structure of NAD and NADH

Nicotinamide adenine dinucleotide has two aromatic groups (Figure 4.7). The adenine part is responsible for the absorption at 260 nm (as in ATP or nucleic acids, cf. Figures 4.5-4.6). The nicotinamide moiety has a specific absorption maximum at 340 nm, but only when protonated (NADH). The very high difference in the absorption between the oxidised and reduced forms (ε NADH,340 = 6220) at this peak makes it a highly useful tool in analytical, enzyme kinetic and medical diagnostic measurements. For example, when measuring ATPase activity, we wish to measure the ADP generated from ATP. However, ATP and ADP cannot be distinguished photometrically—their absorption at 260 nm is the same. In such cases it is common to use a coupled reaction (Figure 4.8). We add enzymes (pyruvate kinase and lactate dehydrogenase) and substrates (PEP, NADH) to the solution so that the oxidation of NADH will be proportional (actually, equimolar) to ATP consumption during the examined reaction. NADH consumption can be readily followed at 340 nm.

Absorption spectrum and molecular structure of NAD+ and NADH

Figure 4.7. Absorption spectra (left) and molecular structure of NAD+ (middle) and NADH (right)

Measuring ATPase activity via a coupled reaction

Figure 4.8. Measuring ATPase activity via a coupled reaction

4.6.4. Absorption spectrum of proteins

Proteinogenic amino acids with an aromatic group in their side chain have a peak in absorption at 280 nm (Figure 4.9). As mentioned earlier, the absorption of amino acids is additive and thus the molar extinction coefficient of proteins can be calculated from their amino acid composition. Non-aromatic amino acids do not absorb at this wavelength, except for the low absorbance of cystine (a pair of cysteines forming a disulfide bond). Absorption values calculated based on amino acid composition generally fall close to values determined experimentally, making them suitable for the determination of protein concentration. The side chains of tryptophan and, to a lesser extent, that of tyrosine are also fluorescent. Tryptophan can be selectively excited at 295 nm, as it can be inferred from the “shoulder” in its absorption spectrum (Figure 4.9).

In the spectrum of proteins, absorption at 220 nm is due to the peptide bonds and the peak at 280 nm is caused by the absorption of aromatic amino acids (Figure 4.10). The latter is better suited for concentration measurements because there are many other materials, including minor impurities in solvents, that exhibit absorption at 220 nm.

Absorption spectra of aromatic amino acids

Figure 4.9. Absorption spectra of aromatic amino acids

Absorption spectrum of a protein

Figure 4.10. Absorption spectrum of a protein

4.6.5. Determination of the purity of DNA and protein samples

The ratio of DNA and protein in biological samples can be estimated from the absorbance of the sample at 260 and 280 nm, resulting from the absorbance of nucleotide bases and aromatic amino acids. In the case of a pure solution of DNA, A260/ A280 = 1.8. The addition of protein to the solution will typically reduce this value. Figure 4.11 shows the absorption spectra of solutions with different DNA to protein ratios.

Determination of the purity of DNA and protein solutions

Figure 4.11. Determination of the purity of DNA and protein solutions