BioPHYSICAL chemistry

Since the rates are first order, the observed rate is given by just the sum

of the rates for all possible decay pathways:

(14.28)

Sometimes the processes other than fluorescence involve mechanisms that

are important to biology, such as photosensing processes like phototaxis,

vision, and photosynthesis (Chapter 20). In these cases, the measurement

of fluorescence provides a sensitive spectroscopic means of probing these

processes.

FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)

Many applications of fluorescence to biological systems make use of energy

transfer between a donor and acceptor. Because such transfer processes are

highly dependent upon the distance, the transfer can serve as a measure

of the donor–acceptor distance. One common method is to perform a

FRET experiment, or fluorescence resonance energy transfer. The electron

donor is excited and, if there is no nearby acceptor, fluorescence from the

donor is observed. By if an acceptor is nearby, then the energy can be

transferred from the donor to the acceptor and fluorescence is observed

from the acceptor. The efficiency of transfer depends upon many factors.

For example, the fluorescence spectrum of the donor should overlap

with the absorption spectrum of the acceptor. In many applications, these

factors are considered to be fixed and the efficiency of transfer depends

on the donor–acceptor distance,rDA, according to:

Efficiency= (14.29)

where rois a parameter that contains the various other factors. The value

of rocan be estimated but is usually determined experimentally with

values of 10 – 60 Å being common.

Measuring fluorescence

Fluorescence can be measured with an emission spectrophotometer. In

this case, a second light source is used for excitation of the sample. This

excitation light is passed through a monochromator before entering the

sample compartment. Excited molecules in the samplefluoresce and the

fluorescence is collected, passed through a second monochromator, and

r

rr

o

oDA+

6

66

kk kobs f i

i

=+∑

306 PART 2 QUANTUM MECHANICS AND SPECTROSCOPY