Index Protein Fluorophores

Basic Definitions and Phenomena of Fluorescence

All molecules absorb light. However, only a relatively low number of molecular species (usually rigid conjugated polyaromatic hydrocarbons or heterocycles) emit light as a result of absorption of light from some other sources. If the emission is immediate or from the electronically excited singlet state, the phenomenon is called fluorescence. Of the 20 amino acids usually found in proteins, the aromatic phenylalanine, tyrosine and tryptophane are the only ones of suffiicient fluorescence intensity to be measured directly in solution. The fluorescence of proteins due to these residues is a highly specific and sensitive tool for studying structure and conformation.

Singlet-Singlet Transition

Fluorescence is the emission of light from a molecule in which an electronically excited state has been populated. The emission of the light is usually in the ultraviolet to visible portion of the spectrum, sometimes in the near-infrared. The phenomenon can be illustrated by a simple electronic state diagram:

The energy levels of the fluorophore are represented by horizontal lines and are grouped in bands. The lowest band is associated with the ground electronic state, S_o. The lowest energy of the excited singlet state is represented by S₁, whereas T₁ is the lowest level of the excited triplet state.

Upon excitation, fluorophores in ground state absorb a photon and jump to higher vibrational energy levels of the electonically excited singlet state, shown as absorption of light, A. The photon of excitation is supplied by an external source, such as an incandescent lamp or a laser. The transition from S_o to higher excited levels of S₁ is responsible for the visible and ultraviolet absorption spectra observed for fluorophores. The absorption of photon is highly specific and it takes place in about 10^-15 second.

Excitation is followed by a return to the lower vibrational levels of the electronically excited state. This relaxation occurs in about a picosecond. The excited state itself exists for a finite time. Typical values of excited-state lifetimes are in the range of nanoseconds, for example 2.6 nsec for tryptophane or 3.6 nsec for tyrosine as tabulated in a subsequent page. The geometry of the fluorochrome in the excited is different from that of the ground state, ie. some bond lengths and bond angles have changed.

From the singlet state, the fluorophore returns to the electronic ground state with the emission of the photon, shown as fluorescence F, but to higher vibrational levels of this state. In fluorescence emission, the spin multiplicities of the ground and excited states are the same. One can measure either a steady state spectrum of emitted light or the actual decay kinetics of emission.

Photoemission is unimolecular. It is a first order process in the concentration of the excited state. The rate constants for fluorescence are typically of the order of 10⁸ s^-1. The energy of the photon that is emitted as the electron decays to the ground state depends on the energy difference between the excited and ground state at the time of emission. The rapid decay of excited vibrational states implies that the state from which the fluorophore decays is independent of the excitation wavelength. However, the state to which the fluorophore decays is not always the lowest vibrational state of the ground state, but it is an equilibrium distribution of vibrational levels. Therefore, the emission spectra of fluorescent molecules show fine structure. The probablility of decay from the excited state to each vibrational level of the ground state is what determines the shape of the fluorescence spectrum.

Triplet-Singlet Transition

During its lifetime, the excited state is subject to a variety of possible interactions with its molecular environment. Processes such as:

• partial dissipation to relaxed singlet excited state
• collisional quenching
• fluorescence energy transfer
• intersystem crossing (see below)

may depopulate the singlet state and thus the energy is lost for fluorescence.

Some fluorophores may leave the excited state via two main processes other than fluorescence, especially at high levels of excitation power. They can either

• irreversibly switch to non-fluorescent species by non-radiative transitions, which is called bleaching and shown as B in the figure.
• or they can undergo intersystem crossing into the triplet state, T₁ from which the fluorophore returns to the ground state by emission of light as phosphorescence, shown as P.

Intersystem crossing occurs when a triplet state lies just below the excited singlet electronic state, ie. there is a near coincidence of two vibrational levels in the excited singlet state and triplet states. In triplet state, the spins of the excited and ground state electrons are no longer paired. This involves the flipping of one of the electron spins so that unpaired electron spin results. Thus, in phosphorescence emissions, the spin multiplicities of the ground and excited states are different. Because the change in spin violates the quantum mechanical spin conservation rules, the decay from the triplet to ground state is very slow and occurs only if there are no other allowed energy paths open. Common rate constants for phosphorescence are in the range 10²-10⁵ s^-1.

There are other mechanisms that can delay the fluorescence emission to very long periods. This can occur if the energy follows some circuitous path in the excited state before returning to the lower vibrational levels of the electronically excited state. Such a delayed fluorescence is different from true phosphorescence which is derived from the triplet state. Delayed fluorescence results from two intersystem crossings, first from the singlet to the triplet, then from the triplet back to the singlet.

Excitation and Emission Spectra

Fluorescent molecules are characterized by their excitation and emission spectra:

• Excitation Spectrum. The emission is viewed at a constant wavelength and the excitation wavelenght is varied. This resembles an absoption spectrum.

• Emission Spectrum. Excitation is at a constant wavelength and the wavelength for viewing emission is varied.

In principle, the energy of the transition from the lowest energy states is the same for both absorption and emission. In reality, however, the average energy of the emitted photon is generally less than the corresponding absorption band. This red shift is due to a change in the local environment of the excited state during its lifetime. The reorganization of solvent dipoles lowers the energy of the excited state (the vibration energy is lost as heat), which causes a red shift in the emission spectra. The magnitude of the red shift depends on the polarity of the solvent. Solvents of higher polarity produce larger red shifts.

The true spectra is obtained by correcting for the wavelength dependent intensity of the light source and for the wavelenght dependent variation of the detector response. These correction are normally made by comparison with the known corrected spectra of a standard, usually quinine in sulfuric acid.

As an example, you will find the corrected excitation and emission spectra of the Green Fluorescent Protein and some of its variants on a subsequent page.

Information about the properties of macromolecules and their interactions with other molecules can be obtained from studies of the fluorescent spectra. There are many environmental factors that affect the fluorescence patterns as well as fluorescent efficiency which is also environmentally dependent. For example, color vision is based on the fact that spectral properties of a common fluorophore, cis-retinal, are altered as a function of protein environment within red, blue, or green opsins.

As another example, you can read about the effects of local environment on the fluorescence of tryptophane in Melittin later.

Quantum Efficiency

The energy of the emitted photon is invariably smaller than that of the absorbed photon because emission is always preceeded by some kind of energy dissipation. For molecules in solution, the excess vibrational energy is lost through collision with the solvent in nonradiative transitions. Thus fluorescence occurs at a longer wavelength than absorbance. The fluorescence quantum yield, Q is a measure of the relative extent to which these processes occur. It is defined as the ratio of the number of fluorescence photons emitted, F to the number of photons absorbed, A:

It is the fraction of fluorophores that decay via the emission of a photon compared to all other processes, ie. the fraction of excited molecules which become deexcited by fluorescence. In other words, Q is the probability that a molecule will fluoresce.

The quantum yield is a parameter which depends on the immediate environment of the fluorophore. Quantum yield values range from 0 to 1. Molecules with larger quantum yields exhibit stronger fluorescence. Absolute measurements of quantum yields and determination of the absolute energy distribution of a fluorescence spectrum are difficult to make experimentally, and are infrequently done in biochemistry. Either of these two parameters requires measuring absolute intensity. The usual method for doing this is by calibrating the spectrofluorometer with a thermophile. This is an instrument whose ability to measure the energy of incident light is wavelength-independent.

In practice, quantum yields are usually determined by comparison of the fluorescence emission of the species of interest with a standard having a known quantum yield. Because the quantum yield is proportional to the area under a fluorescence band in the spectrum, relative quantum yields can be obtained by comparing the peak areas (or heights) under identical excitation conditions.

The relationship between fluorescence intensity, F and molar concentration of the fluorophore, C is given by:

when the illuminating light has a constant wavelength and intensity. F_o is the incident power of the exciting beam, E is the molar absorptivity of the fluorescent species, and b is the sample cell path length. At higher concentrations, significant deviations from this relationship are often observed because of concentration quenching and self absorption. At low to moderate concentrations, this function reduces to a useful linear relationship:

where a is an instrumental factor.

Excited State State Lifetime

The lifetime of the fluorophore is the average value of the time a fluorophore spends in the excited state before it returns to the ground state. Usually, lifetimes of excited states are shorter in fluorescence than in phosphoressence. Fluorescence typically occurs in the range 0.1-100 nanoseconds. Phosphorescence can occur on the timescale of seconds.

By definition, the fluorescence lifetime is the time required by a population of N excited fluorophores to decrease exponentially to N/e by losing excitation energy through fluorescence and other deactivation pathways. Since the intensity of fluorescence is proportional to N, the definition can be written as:

where F and F_o are the intensities at any time t and at t=0. T is the excited state fluorescence lifetime. The definition assumes that the fluorescence arises from a single fluorophore and decays as a single exponential.

The quantum yield is related to the fluorescence lifetime as:

where T_o is the "natural" lifetime, ie. it is the lifetime of the fluorophore in the absence of nonradiative processes. The natural lifetime can be calculated from the spectral properties of the fluorescent species. Relative fluorescence lifetimes can be determined from quantum yield measurements according to:

There are two principal methods to measure fluorescence decay parameters: the pulsed and phase shift methods.

• In the pulsed method, the sample is excited by a short pulse of light which may have a width at half-maximum of 10 nanosecond or less. The fluorescence decay (intensity) is recorded as a function of time from which the lifetime can be calculated by deconvolution, assuming one to three exponentials in the decay curve. The pulse method is also referred to as time-resolved, or time domain method. The decay function is monoexponential for simple fluorophores. Often, however, the decay of fluorescence cannot be described by a single first-order rate process because two or more species are present with different lifetimes. In this case the quantum yield still can be determined from the corrected spectrum, but the relationship between the quantum yield and fluorescence lifetime is complex. In general, the time dependence of the fluorescence decay can be written as a multiexponencial decay curve in terms of an average relaxation time.

• In the phase method, the sample is excited by a sinusoidically modulated light. The finite lifetime of fluorescence causes it to be delayed in phase and to be modulated to a smaller degree. The phase shift is expressed as a phase angle from which the lifetime can be determined using simple relationships between the modulation frequency and the degree of demodulation. The modulation frequencies are of the order of 10-50 MHz for most lifetimes. Shorter lifetimes require higher modulation frequencies upto 2 GHz. Using a single excitation frequency, multiexponential decays cannot be easily resolved. By obtaining phase data at different frequencies, such as in multifrequency phase fluorometry, one can obtain all information needed to resolve complex decay curves. Multifrequency modulation is also referred as frequency domain method. Both the pulse and phase shift methods give essentially identical results.

Index Protein Fluorophores

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