THE AMINO ACIDS

AMINO ACIDS

DEFINITION

Amino acids are basic building block unit of protein. They are group of organic compound containing two functional groups. The amino acids are named because both amino (-NH₂) and carboxyl (-COOH) groups are present in a single molecule. The amino (-NH₂) group is basic and carboxyl (-COOH) group is acidic in nature. They are precursor molecules of many important biological molecules e.g. Neurotransmitter, enzymes, proteins, N-bases etc.

STRUCTURE OF THE AMINO ACIDS

Although more than 300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian pro­teins. Each amino acid (except for proline, which has a secondary amino group) has a carboxyl group, a primary amino group, and a distinctive side chain (“R-group”) bonded to the α-carbon atom. At physiologic pH (approximately pH 7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (– COO^–), and the amino group is protonated (– NH₃⁺). In proteins, almost all of these carboxyl and amino groups are combined through peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation. Thus, it is the nature of the side chains that ultimately dictates the role an amino acid plays in a protein. It is, therefore, useful to classify the amino acids according to the properties of their side chains, that is, whether they are nonpolar (have an even distribution of electrons) or polar (Figures 1).

ABBREVIATIONS AND SYMBOLS FOR COMMONLY OCCURRING AMINO ACIDS

Each amino acid name has an associated three-letter abbreviation and a one-letter symbol. The one-letter codes are deter­mined by the following rules:

1. Unique first letter: If only one amino acid begins with a particular letter, then that letter is used as its symbol. For example, I = isoleucine.

2. Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most com­mon of these amino acids receives this letter as its symbol. For example, glycine is more common than glutamate, so G = glycine.

3. Similar sounding names: Some one-letter symbols sound like the amino acid they represent. For example, F = phenylalanine, or W = tryptophan (“twyptophan” as Elmer Fudd would say).

4. Letter close to initial letter: For the remaining amino acids, a one-letter symbol is assigned that is as close in the alphabet as possi­ble to the initial letter of the amino acid, for example, K = lysine. Furthermore, B is assigned to Asx, signifying either aspartic acid or asparagine, Z is assigned to Glx, signifying either glutamic acid or glutamine, and X is assigned to an unidentified amino acid (Figure 2).

                                    

 Figure 1: Structural features of amino acids

(Shown in their fully protonated form)

   Figure 2: Abbreviations and symbols used for amino acids

CLASSIFICATION OF AMINO ACIDS:

1. Classification of amino acids on the basis of nature of side chains:

A. Amino acids with nonpolar side chains

Each of these amino acids has a nonpolar side chain that does not gain or lose protons or participate in hydrogen or ionic bonds. The side chains of these amino acids can be thought of as “oily” or lipid-like, a property that promotes hydrophobic interactions (Figure 3).

Figure 3: Structures of amino acids with non-polar side chains

B. Polar amino acids with no charge on 'R‘ group:

These amino acids, as such, carry no charge on the 'R‘ group. They however possess groups such as hydroxyl, sulfhydryl and amide and participate in hydrogen bonding of protein structure. The simple amino acid glycine (where R = H) is also considered in this category. The amino acids in this group are glycine, serine, threonine, cysteine, glutamine, asparagine and tyrosine (Figure 4).

Figure 4: Structures of polar amino acids with no charge R groups

C. Amino acids with acidic side chains

The amino acids aspartic and glutamic acid are proton donors. At physiologic pH, the side chains of these amino acids are fully ionized, containing a negatively charged carboxylate group (–COO^–). They are, therefore, called aspartate or glutamate to emphasize that these amino acids are negatively charged at physiologic pH (Figure 5).

Figure 5: Structures of amino acids with acidic side chains

D. Amino acids with basic side chains

The side chains of the basic amino acids accept protons. At physiologic pH the side chains of lysine and arginine are fully ionized and positively charged. In contrast, histidine is weakly basic, and the free amino acid is largely uncharged at physiologic pH. However, when histidine is incorporated into a protein, its side chain can be either positively charged or neutral, depending on the ionic environment provided by the polypeptide chains of the protein. This is an important property of histidine that contributes to the role it plays in the functioning of proteins such as hemoglobin (Figure 6).

Figure 6: Structures of amino acids with basic side chains

2. Nutritional classification of amino acids:

The twenty amino acids are required for the synthesis of variety of proteins, besides other biological functions. However, all these 20 amino acids need not be taken in the diet. Based on the nutritional requirements, amino acids are grouped into two classes essential and nonessential.

A. Essential or indispensable amino acids:

The amino acids which cannot be synthesized by the body and, therefore, need to be supplied through the diet are called essential amino acids. They are required for proper growth and maintenance of the individual. The ten amino acids listed below are essential for humans (and also rats): Arginine, Valine, Histidine, lsoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, and Tryptophan (For remembrance use the code 'PVT TIM HALL'). The two amino acids namely arginine and histidine can be synthesized by adults and not by growing children, hence these are considered as semi-essential amino acids. HA

Thus, 8 amino acids are absolutely essential while 2 are semi-essential.

B. Non-essential or dispensable amino acids:

The body can synthesize about '10 amino acids to meet the biological needs, hence they need not be consumed in the diet. These are glycine, alanine, serine, cysteine, aspartate, asparagi ne, glutamate, glutamine, tyrosine and proline.

3. Amino acid classification based on their metabolic fate

The carbon skeleton of amino acids can serve as a precursor for the synthesis of glucose (glycogenic) or fat (ketogenic) or both. From metabolic view point, amino acids are divided into three groups.

A. Glycogenic amino acids:

These amino acids can serve as precursors for the formation of glucose or glycogen. E.g. alanine, aspartate, glycine, methionine etc.

B. Ketogenic amino acids:

Fat can be synthesized from these amino acids. Two amino acids leucine and lysine are exclusively ketogenic.

C. Glycogenic and ketogenic amino acids:

The four amino acids isoleucine, phenylalanine, tryptophan, tyrosine are precursors for synthesis of glucose as well as fat.

PROPERTIES OF AMINO ACIDS

A. Physical Properties of Amino acids:

The amino acids differ in their physicochemical properties which ultimately determine the characteristics of proteins.

1. Solubility:

            Most of the amino acids are usually soluble in water and insoluble in organic solvents.

2. Melting points:

            Amino acids generally melt at higher temperatures, often above 200°C.

3. Taste:

            Amino acids may be sweet (Gly, Ala, Val), tasteless (Leu) or bitter (Arg, lle). Monosodium glutamate (MSC; ajinomoto) is used as a flavoring agent in food industry, and Chinese foods to increase taste and flavor. Some individuals are intolerant to MSC.

4. Optical property:

The α-carbon of an amino acid is attached to four different chemical groups and is, therefore, a chiral or optically active carbon atom. Glycine is the exception because its α-carbon has two hydrogen substituents and, therefore, is optically inactive. Amino acids that have an asymmetric center at the α-carbon can exist in two forms, designated D (Dextro-dextrorotatory) and L (Levo-levorotatory) that are mirror images of each other. The two forms in each pair are termed stereoisomers, optical isomers, or enantiomers. All amino acids found in proteins are of the L-configuration. However, D-amino acids are found in some antibiotics and in plant and bacterial cell walls (Figure 7).

Figure 7: Mirror imaging of optical isomers of amino acids

5. Amino acids as Ampholytes:

Amino acids contain both acidic (-COOH) and basic (-NH₂) groups. They can donate a proton or accept a proton; hence amino acids are regarded as ampholytes. Zwitterion or dipolar ion: The name zwitter is derived from the German word which means hybrid. Zwitter ion (or dipolar ion) is a hybrid molecule containing positive and negative ionic groups. The amino acids rarely exist in a neutral form with free carboxylic (-COOH) and free amino (-NH₂) groups. In strongly acidic pH (low pH), the amino acid is positively charged (cation) while in strongly alkaline pH (high pH), it is negatively charged (anion). Each amino acid has a characteristic pH (e.g. leucine, pH 6.0) at which it carries both positive and negative charges and exists as zwitterion. Isoelectric pH (symbol pl) is defined as the pH at which a molecule exists as a zwitterion or dipolar ion and carries no net charge. Thus, the molecule is electrically neutral.

Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Recall that acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as “weak” ionize to only a limited extent. The concentration of protons in aqueous solution is expressed as pH, where pH = log 1/[H⁺] or –log [H⁺]. The quantitative relationship between the pH of the solu­tion and concentration of a weak acid (HA) and its conjugate base (A^–) is described by the Henderson-Hasselbalch equation.

B. Chemical Properties of Amino acids:

The general reactions of mostly due to the presence groups namely carboxyl (-COOH) group and amino (-NH₂) group.

Reactions due to -COOH group:

1. Salt formation: Amino acids form salts (-COONa) with bases and esters (-COOR') with alcohols.

2. Decarboxylation: Amino acids undergo decarboxylation to produce corresponding amines.

           

R-CH(NH₃+ )-COO   ®        R-CH₂(NH₃+) + CO₂

This reaction assumes significance in the living cells due to the formation of many biologically important amines. These include histamine, tyramine and g-amino butyric acid (GABA) from the amino acids histidine, tyrosine and glutamate, respectively.

3. Reaction with ammonia: The carboxyl group of dicarboxylic amino acids reacts with NH3 to form amide

                        Aspartic acid + NH₃              ®        Asparagine

                        Glutamic acid + NH₃             ®        Glutamine

Reactions due to -NH₂ group:

4. Acts as bases: The amino groups behave as bases and combine with acids (e.g. HCI) to form salts (-NH₃⁺Cl^-).

5. Reaction with ninhydrin: In the pH range of 4-8, all α- amino acids react with ninhydrin (triketohydrindene hydrate), a powerful oxidizing agent to give a purple colored product (diketohydrin) termed Rhuemann’s purple. All primary amines and ammonia react similarly but without the liberation of carbon dioxide. The imino acids proline and hydroxyproline also react with ninhydrin, but they give a yellow colored complex instead of a purple one. Besides amino acids, other complex structures such as peptides, peptones and proteins also react positively when subjected to the ninhydrin reaction (Note: Proline and hydroxyproline give yellow color with ninhydrin).

6. Color reactions of amino acids: Amino acids can be identified by specific color reactions:

a. Xanthoproteic acid test

Aromatic amino acids, such as Phenyl alanine, tyrosine and tryptophan, respond to this test. In the presence of concentrated nitric acid, the aromatic phenyl ring is nitrated to give yellow colored nitro-derivatives. At alkaline pH, the color changes to orange due to the ionization of the phenolic group.

                    

b. Pauly's diazo Test

This test is specific for the detection of Tryptophan or Histidine. The reagent used for this test contains sulphanilic acid dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in the presence of sodium nitrite and hydrochloric acid results in the formation a diazonium salt. The diazonium salt formed couples with either tyrosine or histidine in alkaline medium to give a red coloured chromogen (azo dye). 

c. Millon's test

Phenolic amino acids such as Tyrosine and its derivatives respond to this test. Compounds with a hydroxybenzene radical react with Millon’s reagent to form a red colored complex. Millon’s reagent is a solution of mercuric sulphate in sulphuric acid.

d. Histidine test

This test was discovered by Knoop. This reaction involves bromination of histidine in acid solution, followed by neutralization of the acid with excess of ammonia.  Heating of alkaline solution develops a blue or violet coloration.

e. Hopkins cole test

This test is specific test for detecting tryptophan. The indole moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric acid to give a purple colored product. Glyoxilic acid is prepared from glacial acetic acid by being exposed to sunlight.

f. Sakaguchi test

Under alkaline condition, α- naphthol (1-hydroxy naphthalene) reacts with a mono-substituted guanidine compound like arginine which upon treatment with hypobromite or hypochlorite produces a characteristic red color.

g. Lead sulphide test

Sulphur containing amino acids, such as cysteine and cystine upon boiling with sodium hydroxide (hot alkali), yield sodium sulphide. This reaction is due to partial conversion of the organic sulphur to inorganic sulphide, which can be detected by precipitating it to lead sulphide, using lead acetate solution.

                                    

h. Folin's McCarthy Sullivan Test

Imino acids such as Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct. Addition to sodium nitroprusside [Na₂Fe(CN)₅NO]  to an alkaline solution of methionine followed by the acidification of the reaction yields a red color. This reaction also forms the basis for the quantitative determination of methionine.  

i. Isatin test

Imino acids such as Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct.

7. Transamination: Transfer of an amino group from an amino acid to a keto acid to form a new amino acid is a very important reaction in amino acid metabolism.

8. Oxidative deamination: The amino acids undergo oxidative deamination to liberate free ammonia.

References:

Lippincott's Bichemistry

&

Satyanarayan's Text Book of Biochemistry

Molecular Luminescence Spectroscopy

MOLECULAR LUMINESCENCE SPECTROSCOPY

Luminescence spectroscopy is a technique which studies the fluorescence, phosphorescence, and chemiluminescence of chemical systems. The analyte or its reaction product needs to be luminescent. The relative luminescence intensity is related to analyte concentration as will be seen shortly.

In favourable cases, luminescence methods are amongst some of the most sensitive and selective of analytical methods available. Detection Limits are as a general rule at ppm levels for absorption spectrophotometry and ppb levels for luminescence methods. Collectively, fluorescence and phosphorescence are known as photoluminescence. A third type of luminescence - Chemiluminescence - is based upon emission of light from an excited species formed as a result of a chemical reaction.

Flourimetry is the most commonly used luminescence method.  Phosphorimetry usually requires at liquid nitrogen temperatures (77K). The terms flourimetry and fluorometry are used interchangeably in the chemical literature. Two wavelength selectors are required filters (in fluorimeters) and monochromators (in Spectrofluorimeters).

Singlet and Triplet States

Electrons in molecular orbitals are paired, according to Pauli Exclusion Principle. When an electron absorbs enough energy it will be excited to a higher energy state; but will keep the orientation of its spin. The molecular electronic state in which electrons are paired is called a singlet transition. On the other hand, the molecular electronic state in which the two electrons are unpaired is called a triplet state. The triplet state is achieved when an electron is transferred from a singlet energy level into a triplet energy level, by a process called intersystem crossing; accompanied by a flip in spin.

In a singlet state, the spins of the two electrons are paired and thus exhibit no magnetic field and called diamagnetic. Diamagnetic molecules, containing paired electron, are neither attracted nor repelled by a magnetic field. On the other hand, molecules in the triplet state have unpaired electrons and are thus paramagnetic which means that they are either repelled or attracted to magnetic fields. The terms singlet and triplet stems from the definition of multiplicity where: Multiplicity = 2S + 1 Where, S is the total spin. The total spin for a singlet state is zero since electrons are paired which gives a multiplicity of one (the term singlet state).  Multiplicity = (2 * 0) + 1 =1. In a triplet state, the total spin is one (the two electrons are unpaired) and the multiplicity is three: Multiplicity = (2 * 1) + 1 = 3 It should also be indicated that the probability of a singlet to triplet transition is much lower than a singlet to singlet transition. Therefore, the intensity of the emission from a triplet state to a singlet state is much lower than emission intensities from a singlet to a singlet state.

Energy Level Diagram for Photoluminescent Molecules

The following diagram represents the main processes taking place in a photoluminescent molecule when it absorbs and emits energy.

Figure 1: Fluorescence emission showing singlet and triplet state

The different processes will be discussed below:

1. Absorption

The absorption of UV-Vis radiation is necessary to excite molecules from the ground state to one of the excited states. Absorption of radiation promotes electrons in chemical bonds to be excited. However, we have seen earlier that not all transitions have the same probability and while certain transitions are practically very important, others are seldom used and are of either no or marginal importance. There are four different types of electronic transitions which can take place in molecules when they absorb UV-Vis radiation. A σ−σ* and a n−σ* are not useful while the n−π* transition requires low energy but the molar absorptivity for this transition is low and transition energy will increase in presence of polar solvents.  The most frequently used transition is the π−π* transition for the following reasons:

a.	The molar absorptivity for the π−π* transition is high allowing sensitive determinations.
b.	The energy required is moderate, far less than dissociation energy.
c.	In presence of the most convenient solvent (water), the energy required for a π−π* transition is usually smaller.

Therefore, best molecules that may show absorption are those with π bonds or preferably aromatic nature. Absorption to higher excited singlet states requires a very short time (in the range of 10^-14s).

2. Vibrational Relaxation

Absorption of radiation will excite molecules to different vibrational levels of the excited state. This process is usually followed by successive vibrational relaxations (VR) as well as internal conversion to lower excited states. In cases where transitions occur to the first excited state, vibrational relaxation to the main excited electronic level will take place and/or an intersystem crossing (ISC) to the triplet state can occur.

3. Fluorescence

After vibrational relaxation to first excited electronic level takes place, a molecule can return to the ground state by emission of a photon, called fluorescence (FL). The fluorescence lifetime is much greater than the absorption time and occurs in the range from 10^-7-10^-9 s. As the lifetime in the excited state is increased, the probability of fluorescence will be decreased since radiationless deactivation processes may take place. However, not all excited molecules can show fluorescence by returning to ground state and most return to ground state by losing excitation energy as heat or through collisions with other molecules or solvent.

4. Internal and External Conversion

Internal conversion (IC) is a radiationless deactivation process whereby excited molecules return to the ground state without emission of a photon. This process lacks rigid understanding but seems to be the most efficient deactivation process in luminescence spectroscopy, since most molecules do not show fluorescence. However, molecules with close electronic energy levels, to the extent that their vibrational energy levels of ground and excited states are overlapped, are believed to cause efficient internal conversion. Internal conversion can result in a phenomenon called predissociation (PD) where an electron relaxes from a higher electronic state to an upper vibrational energy of a lower electronic state. When the vibrational energy is large enough and is greater than the bond synergy, bond rupture occurs in a process called predissociation. Dissociation should be differentiated from predissociation where dissociation involves absorption of high energy so that the molecule is directly promoted to a high energy vibrational level where bond rupture directly occurs. External conversion (EC) is a process whereby excited molecules lose their energy due to collisions with other molecules or by transfer of their energy to solvent or other unexcited molecules. Therefore, external conversion is influenced by temperature, solvent viscosity, as well as solvent composition.

5. Intersystem Crossing

Electrons present at the first excited electronic level can follow one of three choices including emission of a photon to give fluorescence, radiationless deactivation to ground state, or intersystem crossing (ISC). The process of intersystem crossing involves transfer of the electron from an excited singlet to a triplet state. This process can actually take place since the vibrational levels in the singlet and triplet states overlap. However, crossing of the singlet state to the triplet state involves a flip in electron spin in order to satisfy the triplet state. Intersystem crossing is facilitated by presence of nonbonding electrons as well as heavy atoms. The presence of paramagnetic atoms or species also enhances intersystem crossing. An electron in the triplet state can also cross back to the singlet state and can result in a photon as fluorescence but at a much longer time than regular fluorescence. This process is termed delayed fluorescence and has the same characteristics as direct fluorescence except for the large increase in lifetime.

6. Phosphorescence

Electrons crossing the singlet state to the triplet state with a flipped spin can also follow one of three choices including returning to the singlet state (including a flip in spin), relax to ground state by internal or/and external conversion, or lose their energy as a photon (phosphorescence, Ph) and relax to ground state with a second flip in spin to satisfy the singlet ground state. As can be rationalized from the processes involved in collecting phosphorescence photons, this involves an intersystem crossing and two flips in spin. This, in fact, requires a much longer time than fluorescence (10^-4s to up to few s). Therefore, the probability of phosphorescence, and hence the intensity of the phosphorescence spectrum, is very low due to high possibility of radiationless deactivation.

Figure 2: Wavelength pattern of Fluorescence and Phosphorescence

INSTRUMENTATION: Spectrophotometers

1. Light sources Light sources

·                     Low pressure Hg lamp low pressure Hg lamp

·                      254, 302, 313 nm lines 254, 302, 313 nm lines

·                     High pressure xenon arc lamp high pressure xenon arc lamp

·                     Lasers

2. Wavelength selectors Wavelength selectors

Filters

Monochromators

3. Detectors

Photomultipliers

CCD cameras CCD cameras

4. Cells and sample compartments Cells and sample compartments

Quartz cells quartz cells

Light tight compartments to minimize stray light

Note: Optical Diagram of Molecular luminescence is same as that of flourimetry.  

SPECTROFLUOROMETRY

Spectroflouremetry is an Emission phenomenon. It is primarily concerned with electronic and vibrational states. Generally, the species being examined will have a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. In fluorescence spectroscopy, the species is first excited, by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state. The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As molecules may drop down into any of several vibrational levels in the ground state, the emitted photons will have different energies, and thus frequencies causing fluorescence (emission) e.g. In a typical experiment, the different frequencies of fluorescent light emitted by a sample are measured, holding the excitation light at a constant wavelength. This is called an emission spectrum. An excitation spectrum is measured by recording a number of emission spectra using different wavelengths of excitation light.

 

Figure 3: Fluorescent wavelength of Anthracene

Quantum efficiency =

(Note: Independent of exciting wavelength)

Quantum efficiency = Quanta fluoresed / Quanta absorbed

At low concentration;           I_f µ c                           

Spectroflouremetry is most accurate at very low concentration.

Great spectral selectivity=two monochromators

Susceptible to pH, temperature, solvent polarity

Disadvantages                       

1. Quenching               2. Interference

Quenching

The quantum yield of a fluorophore is dependent on several internal and external factors. One of the external factors with practical implications is the presence of a quencher. A quencher molecule decreases the quantum yield of a fluorophore by non-radiating processes. The absorption (excitation) process of the fluorophore is not altered by the presence of a quencher. However, the energy of the excited state is transferred onto the quenching molecules. Two kinds of quenching processes can be distinguished:

•         Dynamic quenching which occurs by collision between the fluorophore in its excited state and the quencher; and

•         Static quenching whereby the quencher forms a complex with the fluorophore. The complex has a different electronic structure compared to the fluorophore alone and returns from the excited state to the ground state by non-radiating processes.

INSTRUMENTATION

                            

Figure 4: Optical Diagram of Spectrofluorimrtry

Figure 5: Optical Diagram of Spectrofluorimetry

Fluorescent radiation is emitted in all directions and the use of second monochromator perpendicular to the emitted radiation is made of this fact to avoid difficulties that may be caused by the transmission of the incident radiation by the sample. By moving the detection system at right angles to the cell only fluorescent radiation is detected. Some instruments are designed to measure front face fluorescence i.e. the radiation that is emitted along a light path at an angle to the incident radiation. Such fluorescence measurements are comparable to reflectance measurements.

Despite the measurement of the emitted radiation by these means, it is still possible for scattered or reflected incident radiation to reach the detector. To prevent this, flourimeter requires a second monochromating system between the sample and the detector. Many simple fluorimeters use filters as both the primary and secondary monochromators but those instruments that use true optical monochromators for both components are known as Spectrofluorimeters. Other instruments incorporate a simple cut-off filter system for the emitted radiation while remaining the optical monochromator for the excitation radiation. Because the wavelengths of both excitation and emission are characteristic of the molecule, it is debatable which monochromator is the most important design of a flourimeter.

The main advantage of fluorescence techniques is their sensitivity and measurements of nanogram (ng) quantities are often possible. The reason for the increased sensitivity of flourimetry over that of molecular absorption spectrophotometer lies in the fact that fluorescence measurements use a non-fluorescent blank solution, which gives a zero or minimal signal from the detector. Absorbance of the incident radiation results in a large response from the detector. The sensitivity of fluoremetric measurements can be increased by using a detector that will accurately measure very small amounts of radiation.

APPLICATIONS:

There are many and highly varied applications for fluorescence despite the fact that relatively few compounds exhibit the phenomenon. The effects of pH, solvent composition and the polarization of fluorescence may all contribute to structural elucidation. Measurement of fluorescence lifetimes can be used to assess rotation correlation coefficients and thus particle sizes. Non-fluorescent compounds are often labeled with fluorescent probes to enable monitoring of molecular events. This is termed extrinsic fluorescence as distinct from intrinsic fluorescence where the native compound exhibits the property. Some fluorescent dyes are sensitive to the presence of metal ions and can thus be used to track changes of these ions in vitro samples, as well as whole cells.

Intrinsic protein fluorescence

·         The important nutrients such as vitamins, Vitamin B, NADH etc. in food products can be determined by the technique as they are intrinsic fluorescent compounds in biological system which is measured even at very low concentration by this technique.

·         Some intrinsic fluorescent compounds such as Hormones and Drugs can be estimated by this technique. It is used in drugs metabolism as well.

·         The use of pesticides can also be estimated by this technique.

·         Proteins possess three inty lorinsic fluorophores: tryptophan, tyrosine and phenylalanine, although the latter has a very low quantum yield and its contribution to protein fluorescence emission is thus negligible. Of the remaining two residues, tyrosine has the lower quantum yield and its fluorescence emission is almost entirely quenched when it becomes ionised, or is located near an amino or carboxyl group, or a tryptophan residue. Intrinsic protein fluorescence is thus usually determined by tryptophan fluorescence which can be selectively excited at 295–305 nm. Excitation at 280 nm excites tyrosine and tryptophan fluorescence and the resulting spectra might therefore contain contributions from both types of residues.

·         The main application for intrinsic protein fluorescence aims at conformational monitoring. We have already mentioned that the fluorescence properties of a fluorophore depend significantly on environmental factors, including solvent, pH, possible quenchers, neighbouring groups, etc. A number of empirical rules can be applied to interpret protein fluorescence spectra:

•         As a fluorophore moves into an environment with less polarity, its emission spectrum exhibits a hypsochromic shift (l_max moves to shorter wavelengths) and the intensity at l_max increases.

•         Fluorophores in a polar environment show a decrease in quantum yield with increasing temperature. In a non-polar environment, there is little change.

•         Tryptophan fluorescence is quenched by neighbouring protonated acidic groups.

·         When interpreting effects observed in fluorescence experiments, one has to consider carefully all possible molecular events. For example, a compound added to a protein solution can cause quenching of tryptophan fluorescence. This could come about by binding of the compound at a site close to the tryptophan (i.e. the residue is surface exposed to a certain degree), or due to a conformational change induced by the compound.

Extrinsic fluorescence

·         Frequently, molecules of interest for biochemical studies are non-fluorescent. In many of these cases, an external fluorophore can be introduced into the system by chemical coupling or non-covalent binding. Three criteria must be met by fluorophores in this context.

                                i.            Firstly, it must not affect the mechanistic properties of the system under investigation.

                              ii.            Secondly, its fluorescence emission needs to be sensitive to environmental conditions in order to enable monitoring of the molecular events.

                            iii.            And lastly, the fluorophore must be tightly bound at a unique location.

                                                                                              

·         A common non-conjugating extrinsic chromophore for proteins is 1-anilino-8-naphthalene sulphonate (ANS) which emits only weak fluorescence in polar environment, i.e. in aqueous solution. However, in non-polar environment, e.g. when bound to hydrophobic patches on proteins, its fluorescence emission is significantly increased and the spectrum shows a hypsochromic shift; l_max shifts from 475 nm to 450 nm. ANS is thus a valuable tool for assessment of the degree of non-polarity. It can also be used in competition assays to monitor binding of ligands and prosthetic groups.

·         Reagents such as fluorescamine, o-phthalaldehyde or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate have been very popular conjugating agents used to derivatise amino acids for analysis. O-Phthalaldehyde, for example, is a non-fluorescent compound that reacts with primary amines and b-mercaptoethanol to yield a highly sensitive fluorophore.

·         Metal-chelating compounds with fluorescent properties are useful tools for a variety of assays, including monitoring of metal homeostasis in cells. Widely used probes for calcium are the chelators Fura-2, Indo-1 and Quin-1. Since the chemistry of such compounds is based on metal chelation, cross-reactivity of the probes with other metal ions is possible.

·         The intrinsic fluorescence of nucleic acids is very weak and the required excitation wavelengths are too far in the UV region to be useful for practical applications. Numerous extrinsic fluorescent probes spontaneously bind to DNA and display enhanced emission. While in earlier days ethidium bromide was one of the most widely used dyes for this application, it has nowadays been replaced by SYBRGreen, as the latter probe poses fewer hazards for health and environment and has no teratogenic properties like ethidium bromide. These probes bind DNA by intercalation of the planar aromatic ring systems between the base pairs of double-helical DNA. Their fluorescence emission in water is very weak and increases about 30-fold upon binding to DNA.