Measurements of Absolute Values in Biochemical Fluorescence Spectroscopy

Fluorescence spectroscopy is an important tool of the bioche mist studying the structure and interac tion s of prote ins and nucle ic acid s. The four bas ic quantities to measure accurately are: 1. s pec tra (co rrected excitation and emiss ion). 2. quantum yields (0), 3. fluorescence decay characte ri stics, and 4_ poLarization_ Co mmercially availab le instruments, with little modification, can be used to obtain these measureme nts, but the bioc he mi st in this fi eld is ve ry de pendent on the accuracy of measure ments of substances he uses as s tandards. Confusion arises from disagree ment betwee n re ported values for s tandards which may be used to ca ubrate a detector sys te m to obtain quantum yields, or to set up Ijfe time and polarization photometers. For instance, the prot ein chemist is fond of using tryptophan and quinine as quantum yield standards, but Q for tryptophan has been various ly reported as 0.13 and 0.20, and Q values for quinine bisulfate range from 0.4 to 0.7. The bioche mist should also be aware of the proble ms inhe re nt in the use of comme ri ca lly avai lable instrume nts in absolute measurements, as we ll as the s pecial complications arisin g in co mplex bioc he mical systems where the flu orescence is hete rogeneous.