Principles and Applications of Fluorescence Spectroscopy

Fluorescence spectroscopy is an important investigational tool in many areas of analytical science, due to its extremely high sensitivity and selectivity. With many uses across a broad range of chemical, biochemical and medical research, it has become an essential investigational technique allowing detailed, real-time observation of the structure and dynamics of intact biological systems with extremely high resolution. It is particularly heavily used in the pharmaceutical industry where it has almost completely replaced radiochemical labelling. Principles and Applications of Fluorescence Spectroscopy gives the student and new user the essential information to help them to understand and use the technique confidently in their research. By integrating the treatment of absorption and fluorescence, the student is shown how fluorescence phenomena arise and how these can be used to probe a range of analytical problems. A key element of the book is the inclusion of practical laboratory experiments that illustrate the fundamental points and applications of the technique. Straightforward overview of absorption and fluorescence shows the student how the phenomenon arises and how it can be used in the course of their research. Highly practical approach shows non-specialists how to use the technique to investigate chemical and biochemical problems and generate sophisticated results. Contains many easy to perform laboratory experiments to reinforce the clear explanations.

Table of Contents:
1 Absorption Spectroscopy Theory 1 1.1 Introduction 1 1.2 Characteristics of an Absorption Spectrum 2 1.3 Beer–Lambert–Bouguer Law 4 1.4 Effect of the Environment on Absorption Spectra 6 References 11 2 Determination of the Calcofluor White Molar Extinction Coefficient Value in the Absence and Presence of α1-Acid Glycoprotein 13 2.1 Introduction 13 2.2 Biological Material Used 13 2.2.1 Calcofluor White 13 2.2.2 α1-Acid glycoprotein 13 2.3 Experiments 16 2.3.1 Absorption spectrum of Calcofluor free in PBS buffer 16 2.3.2 Determination of ε. value of Calcofluor White free in PBS buffer 16 2.3.3 Determination of Calcofluor White ε. value in the presence of α1-acid glycoprotein 16 2.4 Solution 17 References 19 3 Determination of Kinetic Parameters of Lactate Dehydrogenase 21 3.1 Objective of the Experiment 21 3.2 Absorption Spectrum of NADH 21 3.3 Absorption Spectrum of LDH 22 3.4 Enzymatic Activity of LDH 22 3.5 Kinetic Parameters 22 3.6 Data and Results 22 3.6.1 Determination of enzyme activity 23 3.6.2 Determination of kinetic parameters 23 3.7 Introduction to Kinetics and the Michaelis–Menten Equation 26 3.7.1 Definitions 26 3.7.2 Reaction rates 26 References 32 4 Hydrolysis of p-Nitrophenyl-β-D-Galactoside with β-Galactosidase from E. coli 34 4.1 Introduction 34 4.2 Solutions to be Prepared 35 4.3 First-day Experiments 35 4.3.1 Absorption spectrum of PNP 35 4.3.2 Absorption of PNP as a function of pH 36 4.3.3 Internal calibration of PNP 37 4.3.4 Determination of β-galactosidase optimal pH 39 4.3.5 Determination of β-galactosidase optimal temperature 40 4.4 Second-day Experiments 40 4.4.1 Kinetics of p-nitrophenyl-β-D-galactoside hydrolysis with β-galactosidase 40 4.4.2 Determination of the β-galactosidase concentration in the test tube 42 4.5 Third-day Experiments 44 4.5.1 Determination of Km and Vmax of β-galactosidase 44 4.5.2 Inhibiton of hydrolysis kinetics of p-nitrophenyl-β-D-galactoside 45 4.6 Fourth-day Experiments 47 4.6.1 Effect of guanidine chloride concentration on β-galactosidase activity 47 4.6.2 OD variation with guanidine chloride 48 4.6.3 Mathematical derivation of Keq 48 4.6.4 Definition of the standard Gibbs free energy, ΔG◦’ 51 4.6.5 Relation between ΔG◦’ and  ΔG’ 51 4.6.6 Relation between ΔG◦’ and Keq 52 4.6.7 Effect of guanidine chloride on hydrolysis kinetics of p-nitrophenyl-β-D-galactoside 56 References 57 5 Starch Hydrolysis by Amylase 59 5.1 Objectives 59 5.2 Introduction 59 5.3 Materials 61 5.4 Procedures and Experiments 61 5.4.1 Preparation of a 20 g l−1starch solution 61 5.4.2 Calibration curve for starch concentration 61 5.4.3 Calibration curve for sugar concentration 63 5.4.4 Effect of pH 64 5.4.5 Temperature effect 66 5.4.6 Effect of heat treatment at 90◦C 69 5.4.7 Kinetics of starch hydrolysis 70 5.4.8 Effect of inhibitor (CuCl2) on the amylase activity 73 5.4.9 Effect of amylase concentration 73 5.4.10 Complement experiments that can be performed 77 5.4.11 Notes 77 References 78 6 Determination of the pK of a Dye 79 6.1 Definition of pK 79 6.2 Spectrophotometric Determination of pK 79 6.3 Determination of the pK of 4-Methyl-2-Nitrophenol 81 6.3.1 Experimental procedure 81 6.3.2 Solution 83 References 87 7 Fluorescence Spectroscopy Principles 88 7.1 Jablonski Diagram or Diagram of Electronic Transitions 88 7.2 Fluorescence Spectral Properties 91 7.2.1 General features 91 7.2.2 Stokes shift 93 7.2.3 Relationship between the emission spectrum and excitation wavelength 94 7.2.4 Inner filter effect 95 7.2.5 Fluorescence excitation spectrum 95 7.2.6 Mirror–image rule 95 7.2.7 Fluorescence lifetime 96 7.2.8 Fluorescence quantum yield 101 7.2.9 Fluorescence and light diffusion 102 7.3 Fluorophore Structures and Properties 102 7.3.1 Aromatic amino acids 104 7.3.2 Cofactors 108 7.3.3 Extrinsinc fluorophores 108 7.4 Polarity and Viscosity Effect on Quantum Yield and Emission Maximum Position 111 References 113 8 Effect of the Structure and the Environment of a Fluorophore on Its Absorption and Fluorescence Spectra 115 Experiments 115 Questions 117 Answers 119 Reference 123 9 Fluorophore Characterization and Importance in Biology 124 9.1 Experiment 1. Quantitative Determination of Tryptophan in Proteins in 6 M Guanidine 124 9.1.1 Introduction 124 9.1.2 Principle 124 9.1.3 Experiment 125 9.1.4 Results obtained with cytochrome b2 core 126 9.2 Experiment 2. Effect of the Inner Filter Effect on Fluorescence Data 127 9.2.1 Objective of the experiment 127 9.2.2 Experiment 127 9.2.3 Results 128 9.3 Experiment 3. Theoretical Spectral Resolution of Two Emitting Fluorophores Within a Mixture 130 9.3.1 Objective of the experiment 130 9.3.2 Results 132 9.4 Experiment 4. Determination of Melting Temperature of Triglycerides in Skimmed Milk Using Vitamin A Fluorescence 134 9.4.1 Introduction 134 9.4.2 Experiment to conduct 136 9.4.3 Results 136 References 138 10 Fluorescence Quenching 139 10.1 Introduction 139 10.2 Collisional Quenching: the Stern–Volmer Relation 140 10.3 Different Types of Dynamic Quenching 145 10.4 Static Quenching 147 10.4.1 Theory 147 10.5 Thermal Intensity Quenching 154 References 159 11 Fluorescence Polarization 160 11.1 Definition 160 11.2 Fluorescence Depolarization 162 11.2.1 Principles and applications 162 11.3 Fluorescence Anisotropy Decay Time 165 11.4 Depolarization and Energy Transfer 166 References 167 12 Interaction Between Ethidium Bromide and DNA 168 12.1 Objective of the Experiment 168 12.2 DNA Extraction from Calf Thymus or Herring Sperm 168 12.2.1 Destruction of cellular structure 168 12.2.2 DNA extraction 168 12.2.3 DNA purification 169 12.2.4 Absorption spectrum of DNA 169 12.3 Ethidium Bromide Titration with Herring DNA 169 12.4 Results Obtained with Herring DNA 170 12.4.1 Absorption and emission spectra 170 12.4.2 Analysis and interpretation of the results 173 12.5 Polarization Measurements 177 12.6 Results Obtained with Calf Thymus DNA 179 12.7 Temperature Effect on Fluorescence of the Ethidium Bromide–DNA Complex 180 References 182 13 Lens culinaris Agglutinin: Dynamics and Binding Studies 184 13.1 Experiment 1. Studies on the Accessibility of I− to a Fluorophore: Quenching of Fluorescein Fluorescence with KI 184 13.1.1 Objective of the experiment 184 13.1.2 Experiment 184 13.1.3 Results 185 13.2 Experiment 2. Measurement of Rotational Correlation Time of Fluorescein Bound to LCA with Polarization Studies 187 13.2.1 Objective of the work 187 13.2.2 Polarization studies as a function of temperature 187 13.2.3 Polarization studies as a function of sucrose at 20◦C 187 13.2.4 Results 189 13.3 Experiment 3. Role of α-L-fucose in the Stability of Lectin–Glycoproteins Complexes 190 13.3.1 Introduction 190 13.3.2 Binding studies 191 13.3.3 Results 192 References 196 14 Förster Energy Transfer 197 14.1 Principles and Applications 197 14.2 Energy-transfer Parameters 202 14.3 Bioluminescence Resonance Energy Transfer 204 References 208 15 Binding of TNS on Bovine Serum Albumin at pH 3 and pH 7 210 15.1 Objectives 210 15.2 Experiments 210 15.2.1 Fluorescence emission spectra of TNS–BSA at pH 3 and 7 210 15.2.2 Titration of BSA with TNS at pH 3 and 7 210 15.2.3 Measurement of energy transfer efficiency from Trp residues to TNS 211 15.2.4 Interaction between free Trp in solution and TNS 211 15.3 Results 211 16 Comet Test for Environmental Genotoxicity Evaluation: A Fluorescence Microscopy Application 220 16.1 Principle of the Comet Test 220 16.2 DNA Structure 220 16.3 DNA Reparation 221 16.4 Polycyclic Aromatic Hydrocarbons 222 16.5 Reactive Oxygen Species 223 16.6 Causes of DNA Damage and Biological Consequences 224 16.7 Types of DNA Lesions 225 16.7.1 Induction of abasic sites, AP, apurinic, or apyrimidinic 225 16.7.2 Base modification 225 16.7.3 DNA adducts 225 16.7.4 Simple and double-stranded breaks 225 16.8 Principle of Fluorescence Microscopy 225 16.9 Comet Test 227 16.9.1 Experimental protocol 227 16.9.2 Nature of damage revealed with the Comet test 227 16.9.3 Advantages and limits of the method 227 16.9.4 Result expression 231 References 231 17 Questions and Exercises 232 17.1 Questions 232 17.1.1 Questions with shorts answers 232 17.1.2 Find the error 232 17.1.3 Explain 233 17.1.4 Exercises 234 17.2 Solutions 241 17.2.1 Questions with short answers 241 17.2.2 Find the error 243 17.2.3 Explain 243 17.2.4 Exercises solutions 244 Index 253

About the Author :
Jihad-René Albani is Associate Professor and head of the Laboratoire de Biophysique Moléculaire at the Université des Sciences et Technologies de Lille, France.