Probing nonenzymatic glycation of proteins by deep ultraviolet light emitting diode induced autofluorescence

The suitability of deep-UV-LED (285 nm) as an excitation source to induce autofluorescence in nonenzymatically glycated proteins has been reported for the first time in this study. Non-enzymatically glycated proteins show high autofluorescence when excited with deep-UV light, i.e., deep-UV-induced autofluorescence (deep-UV-IAF). Multiple autofluorescence peaks of nonenzymatically glycated proteins between 300 and 600 nm when excited using the deep-UV-LED revealed structural and biochemical modifications. The partial unfolding of proteins in which Tryptophan (Trp) is either absent (e.g., RibonucleaseA) or the emission maxima of Trp is insensitive to nonenzymatic glycation (e.g., Human Serum Albumin and Bovine Serum Albumin) were elucidated using their Tyrosine (Tyr) emission (lambda(em) = similar to 320 nm). Also, the deep-UV-LED-induced autofluorescence (deep-UV-LED-IAF) is shown to detect and track a wide range of clinically relevant advanced glycation end-products (AGEs) such as Pentosidine (lambda(em) = similar to 380 nm), Argpyrimidine (lambda(em) = similar to 395 nm), Vesperlysine C (lambda(em) = similar to 405 nm), Vesperlysine A/B (lambda(em) = similar to 440 nm), Crossline (lambda(em) = similar to 480 nm), and Arginine derived AGEs (lambda(em) = similar to 525 nm) which is also supported by the chemometric analysis (PCA). The relevance of Trp/Tyr makeup of proteins in tracking AGEs using deep-UV-IAF has been carefully examined with proteins such as RibonucleaseA (RNaseA:zero Trp and six Tyr), Human Serum Albumin (HSA: one Trp and eighteen Tyr), Bovine Serum Albumin (BSA: two Trp and twenty Tyr) and Hemoglobin (Hb: four Trp and twelve Tyr). The Molecular Dynamic (MD) simulation revealed a high root-mean-square deviation (RMSD: 4.6 angstrom) and an increased average distance between Tyr residues and Trp214 (23.2 angstrom) in methylglyoxal (MG) treated HSA. This confirms the MG-induced protein unfolding and decreased fluorescence resonance energy transfer (FRET) from Tyr to Trp (Tyr -> Trp). The study also used systematic steady-state and time-resolved fluorescence (TRF) to explain the sudden decrease in AGEs specific fluorescence intensity and lifetime at higher concentrations of MG due to inter-AGEs FRET.

Automated summary

The study reports the suitability of deep-UV-LED as an excitation source for inducing autofluorescence in nonenzymatically glycated proteins. Deep-UV-induced autofluorescence of these proteins reveals structural and biochemical modifications, and can detect various clinically relevant advanced glycation end-products.