Chapter 7: Thesis summary and future perspectives
1.5 Chromophores in Proteins
The phenomena of UV light absorption by proteins has been proposed as a structural probe since the early days of molecular biology (Zaccai et al., 2017). But, since absorption of water itself is strong below 170 nm (Quickenden and Irvin, 1980), absorption studies on biological macromolecules are restricted to above 170 nm. In general, three classes of chromophores are predominant in proteins.
1.5.1 Peptide bond
In proteins, the electronic transitions involving peptide bond occur in the far-UV region.
The electrons of peptide bonds are delocalized over the N, C, and O atoms. Also, near the O atom, a nonbonding, n-orbital electron is present. Such electronic transitions show two distinct peaks: (Hunt and Simpson, 1953) one strong peak (arise due to -* transitions) at 190 nm (Ԑ = 7000 M-1cm-1) while another peak of a weaker intensity (arise due to n-*
transitions) at about 210–220 nm (Ԑ = 100 M-1cm-1) (Ham and Platt, 1952). The n-*
transition is symmetry forbidden, thus having weaker intensity and it just forms a shoulder
on to the -* transition peak. At around 175 nm, a third transition can be observed which constitutes an n-σ* transition.
The secondary structural changes in protein can influence the absorption of peptide bond.
Both poly-L-glutamic acid (Imahori and Tanaka, 1959) and poly-L-lysine shows changes in absorption intensities with conformational changes. The random coil or -conformation of peptide shows increased absorption intensity as compared to -helical conformation (Rosenheck and Doty, 1961).
1.5.2 Aromatic amino acids
All charged amino acids (Asp, Glu, Asn, Gln, Arg and His) have relatively weak electronic transitions at around 210 nm because they are masked by the more intense absorption of peptide bond in proteins. The absorption of the peptide bond in the region of 220 nm has been used in the quantification of proteins but it interferes considerably with many other compounds at this wavelength and thus subsequently cannot be justified as a gold standard for estimation. Thus, only the absorptions involving side chain optical properties occurring at wavelengths longer than 230 nm are of importance, where peptide absorption is insignificant. Only the aromatic amino acids: Trp, Tyr and Phe absorb significantly in the near-UV region because of the aromatic moiety and thus serve the limitation stated earlier.
Among the three aromatic amino acids, Trp shows the strongest absorption in the near-UV region (Ԑ = 5,600 M-1cm-1 at 280 nm) (Bent and Hayon, 1975b). This complex absorption arises from the indole side chain of Trp. It basically comprises two major peaks; one near 220 nm (Ԑ = 36,000 M-1 cm-1) and another at 280 nm (Ԑ = 5,600 M-1 cm-1) (Creed, 1984a).
At least two independent electronic transitions are known to comprise the spectra of Trp in the 260-310 nm, with -* transition being one of them.
Tyrosine is another aromatic residue with appreciable absorption in the near-UV region and its electronic transitions occur at 275 nm (Ԑ = 1400 M-1cm-1) and 222 nm (Ԑ = 9000 M-1cm-
1) (Longworth et al., 1971), the 275 nm absorption band arises due to -* transition. At alkaline pH, the tyrosine side chain OH group deprotonates (pKa = 10.07) (Grinspan et al., 1966) and the resulting tyrosinate ion (Tyr-O-) shows a red-shift in the absorption profile as
compared to tyrosine, with absorption maxima at 240 nm (Ԑ = 1100 M-1cm-1) and 290 nm (Ԑ = 2300 M-1cm-1) (Antosiewicz and Shugar, 2016a; Creed, 1984b). Titration of Tyr residues in proteins or the separate determination of Tyr and Trp contributions to an observed absorption spectrum uses this peculiar pH sensitivity of Tyrosine absorption profile (Antosiewicz and Shugar, 2016b). As far as the pH sensitivity is concerned, the Tyr absorbance is more sensitive than that of Trp.
Phe shows two absorption bands: one low intensity absorption band (Bent and Hayon, 1975a) (-* transition) around 257 nm (Ԑ = 200 M-1cm-1) (Wetlaufer, 1963) and another band at around 205 nm (Ԑ = 9600 M-1cm-1). Change in pH has no such impact in the spectrum of Phe (Longworth et al., 1971). Apart from these three aromatic amino acids, Cys and Met, which are basically sulphur containing amino acids show low absorption bands in 230-240 nm range (Wetlaufer, 1963). But, these transitions interfere with the absorption band from peptide bond and are not easily measurable in proteins. However, the disulphides (cystine) have longer-wavelength transitions with max values between 250-270 nm (Ԑ = 300 M-1cm-1) as compared to cysteine (Otey and Greenstein, 1954) and occurs in high proportions in many proteins. Thus, the disulphides absorption spectra is also taken into account for the near- UV absorption in proteins.
The imidazole group of His in its side chain also absorbs appreciably between 185- 220 nm (Ԑ = 6000 M-1 cm-1 at 212 nm) (Wetlaufer, 1963) but not as much as the other amino acids which absorb much more strongly in this region.
1.5.3 Prosthetic groups and Co-Enzymes
Various proteins possess tightly bound non-protein part namely, prosthetic groups (For e.g.
heme, flavin, carotenoid) which are vital for many biological activity. Such metal–protein complex (for e.g. Azurin, Xanthine oxidase) along with many important coenzymes of proteins such as FAD, NADH and NAD+ displays strong UV-Vis absorption. FAD absorbs at 450 nm (Ԑ = 11,300 M-1cm-1) (Aliverti et al., 1999), NADH at 340 nm (Ԑ = 6220 M−1cm−1) and NAD+ at 259 nm (Ԑ = 16,900 M−1cm−1) (Dawson et al., 1969). Heme, which contains a porphyrin ring shows a very intense absorption in the visible region at 404 nm (Ԑ
= 1,70,000 M−1cm−1) (Karnaukhova et al., 2014), and thus their absorption spectra can be
monitored to follow kinetics of the protein. The light absorption properties of chromophore in proteins are of direct biological relevance as in retinal in vision and chlorophyll in photosynthesis. Apart from the peptide bond, aromatic amino acids, disulphide bond and the prosthetic groups, the absorption spectrum of a protein is expected to be optically negligible beyond 315 nm and practically no absorption signals in this spectral region is expected.