Principles and Applications of Fluorescence Spectroscopy

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 K_m and V_max 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 K_eq 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 K_eq 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