In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220–230 nm. In the last 10 years we have observed a worrying increase in the number of articles claiming that this fluorescence originates from the protein backbone, contrary to the established knowledge that UV protein emission is due to aromatic amino acids only. Overall, our data clearly demonstrate that the observed emission upon excitation at 220–230 nm is due to the excitation of Tyr and/or Trp, with subsequent emission from the lowest excited state (i.e. the same as obtained with 280 nm excitation) in agreement with Kasha's rule. Therefore, this fluorescence peak does not provide any information on backbone conformation, but simply reports on the local environment around the aromatic side chains, just as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence as an established fact. We hope the current paper helps counter this new situation and leads to a reassessment of those papers that make this erroneous claim.

Interaction of a pharmacologically important flavonoid, pinostrobin (PS) with the major transport protein of human blood circulation, human serum albumin (HSA) has been examined using a multitude of spectroscopic techniques and molecular docking studies. Analysis of the fluorescence quenching data showed a moderate binding affinity (1.03 × 10⁵ M⁻¹ at 25°C) between PS and HSA with a 1∶1 stoichiometry. Thermodynamic analysis of the binding data (ΔS = +44.06 J mol⁻¹ K⁻¹ and ΔH = −15.48 kJ mol⁻¹) and molecular simulation results suggested the involvement of hydrophobic and van der Waals forces, as well as hydrogen bonding in the complex formation. Both secondary and tertiary structural perturbations in HSA were observed upon PS binding, as revealed by intrinsic, synchronous, and three-dimensional fluorescence results. Far-UV circular dichroism data revealed increased thermal stability of the protein upon complexation with PS. Competitive drug displacement results suggested the binding site of PS on HSA as Sudlow’s site I, located at subdomain IIA, and was well supported by the molecular modelling data.

Binding of gefitinib (GEF), a promising anticancer drug to human serum albumin (HSA), the major transport protein in blood circulation was investigated using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as computational modeling. Fluorescence quenching of HSA upon GEF addition was found to be a static quenching process, as revealed from the decreasing trend of the Stern–Volmer quenching constant with increasing temperature as well as UV-vis absorption spectral results. Fluorescence quenching titration results demonstrated moderate binding affinity with the binding constant, K_a value as 1.70 × 10⁴ M⁻¹ between GEF and HSA at 15 °C. Thermodynamic data (ΔH = −7.74 kJ mol⁻¹ and ΔS = +54.06 J mol⁻¹ K⁻¹) suggested participation of both hydrophobic interactions and hydrogen bonds in stabilizing the GEF–HSA complex, which was further supported by computational modeling results. The far-UV and the near-UV CD spectra showed secondary and tertiary structural changes in HSA, whereas three-dimensional fluorescence spectral results indicated microenvironmental perturbations around protein fluorophores upon GEF binding. Binding of GEF to HSA offered significant protection to the protein against thermal destabilization. Competitive site-marker displacement results along with computational modeling analysis suggested a preferred location of the GEF binding site as site III, located in subdomain IB of HSA. Some common metal ions have been found to interfere with GEF–HSA interaction.