Solution-state characteristics of the ultraviolet A-induced visible fluorescence from proteins.
In response to illumination by ultraviolet-A (UV-A) light, proteins in solid form are now known to display a visible blue fluorescence, ostensibly on account of excitation transitions of loosely-held electrons within peptide bond orbitals engaged in hydrogen bonding. Because the CO and NH atom groups in peptide bonds are generally engaged in extensive hydrogen bonding in globular proteins even in aqueous solution, one could argue that proteins in solution must also display this novel blue fluorescence. Here, using high concentrations to enhance detectability, two globular proteins, gamma-crystallin, and lysozyme, are shown to fluoresce visibly, exhibiting: (a) two excitation maxima, at approximately 315 nm and approximately 385 nm, (b) maximal emission at 425 nm in 100 mg/ml lysozyme and 465 nm in 100 mg/ml gamma-crystallin, (c) a time-resolved emission decay that is best fitted by a sum of three exponentials with lifetimes of 3.14, 0.46, and 9.08 ns, respectively, and comparable relative amplitudes of around 30-40 percent each, and (d) a weak CD spectrum displaying a positive band at approximately 385 nm and a negative band at approximately 465 nm. While the wavelength of maximal emission ((em)lambda(max)) in lysozyme is the same for all protein concentrations, the (em)lambda(max) of gamma-crystallin varies with protein concentration, suggesting a certain degree of conformation dependence.
Guptasarma
Purnananda
P
Protein Science & Engineering Division, Institute of Microbial Technology, Council of Scientific & Industrial Research, Chandigarh 160036, India. pg@imtech.res.in
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D005453:Fluorescence; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D009113:Muramidase / Q000737:chemistry / Q000528:radiation effects; D011506:Proteins / Q000737:chemistry* / Q000528:radiation effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000528:radiation effects; D012996:Solutions; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays; D038222:gamma-Crystallins / Q000737:chemistry / Q000528:radiation effects
D011506:Proteins; D011994:Recombinant Proteins; D012996:Solutions; D038222:gamma-Crystallins; D009113:Muramidase