Techniques for Measuring Fluorescence and Phosphorescence of Biological Materials*

Abstract

Abstract— Techniques and instruments for measuring the luminescence, fluorescence. and phosphorescence of proteins and nucleic acids are described. Emphasis is placed on the principles of the instruments and the procedures which are necessary to measure quantitatively the luminescence from biological macromolecules at 77°K. Examples are given of the effect of denaturation on luminescence of both proteins and nucleic acids. on ways in which the luminescence can be associated with particular constituents, and finally on the influence that electronic energy transfer has on the excitation spectra of protein luminescences. These examples typify the relationship of luminescence spectra to the conformation and constitution of macromolecules.

Correction of right-angle fluorescence measurements for the absorption of excitation radiation

“…It should be noted at this point that Equation 20 is identical except in form with an equation presented by Parker and Barnes (17) for the correction factor 2.303A(jCi -x2) nrAx--10' * However, AIw=dwAW (12) and solving Equation 11for dlw/dw, and substituting into Equation 12, yields (-AIw)=2.3Iw(At + Ac)Aw (13) Substituting back in Equation 10gives Fw = 2.3KIwAtAw (14) This produces from Equation 9Fco = 2.3KA^Aw = 2.3KAt(wl -w2) (15) An equally appealing alternate approach would be to define corrected fluorescence as the observed fluorescence divided by the average intensity of the excitation beam across the window from to w2. Then…”

“…This effect has been noted by several investigators (2,14,15). To minimize it, the general recommendation of working with extremely dilute solutions has been made.…”

“…The primary absorption processes, which are independent of all of the other photophysical and instrumental variables, reduce the intensity of the excitation radiation and, as a result, reduce the amount of the observed fluorescence. This effect has been noted by several investigators (2,14,15). To minimize it, the general recommendation of working with extremely dilute solutions has been made.…”

Kinetics of precursor cleavage at the dibasic sites

“…The fact that the shapes of absorption and emission spectra are conserved suggests that these residues were in contact with the aqueous solvent without any conformational change of pro‐OT/Np(7–15) peptides (see CD data). However, as observed for other peptides [16–18], their fluorescence quantum yields are smaller than that of the free residues. These lower values result mainly from peptide bonds and inductive effects as proton transfer and electron‐withdrawing [19–21].…”

Triplet-Triplet Energy Transfer in α-Trypsin

“…The phosphorescence of compounds can usually only be demonstrated in glasses at low temperatures, since 02 quenches the triplet state in fluid media at room temperatures (15). A sufficient amount of glycerol was therefore added to the enzyme solution so that an optically transparent glass could be obtained at 770K.…”