Carbonyl-based blue autofluorescence of proteins and amino acids

Abstract

Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF’s sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA.

Towards Fingermark Dating: A Raman Spectroscopy Proof‐of‐Concept Study

“…At longer times, when most of the Trp has oxidized, the slower decay in ratio may be related to other degradation reactions, for example those that result in protein carbonyl groups. In support of that is the recent discovery of carbonyl‐based intrinsic protein fluorescence accompanied by absorbance at 360 nm and emission spectra centered at 430–450 nm (depending on the protein identity) …”

Anomalous fluorescence of white hair compared to other unpigmented keratin fibres

“…Note the very strong tryptophan feature near (290, 330) for wool, rabbit hair, cashmere and mink fur that is absent in natural white hair. The visible feature at (380, 450) is present in all cases and is in an identical position to the protein deep‐blue autofluorescence (dbAF) observed in a range of proteins and amino acids [9]. Silk fibroin is included for comparison, contour interval 200 units, range 3000.…”

“…As mentioned in the introduction, recent studies have proposed that visible fluorescence in proteins excited at 360–380 nm and emitting at 450 nm, which is referred to as deep‐blue autofluorescence (dbAF), is due to carbonyl groups [9]. This seems to be one example of a more general phenomenon called non‐traditional intrinsic fluorescence (NTIF) [33], which includes polymers, aliphatic amines, dendrimers, as well as amino acids and proteins.…”

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“…The inherent fluorescence emission of proteins is caused by three aromatic amino acid residues: tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). The Trp residue dominantly is responsible for observed inherent protein fluorescence, because of the low quantum yield of the Phe and Tyr residues . Trp fluorescence properties that come from the indole group in its structure are the most sensitive to its environment (polarity of its micro‐environment, noncovalent interactions, hydrogen bonding and denaturation) and can change upon interaction with various ligands (drugs or small molecules) and protein folding/unfolding.…”