Principles and Applications of Fluorescence Spectroscopy -- Contents -- 1 Absorption Spectroscopy Theory -- 1.1 Introduction -- 1.2 Characteristics of an Absorption Spectrum -- 1.3 Beer-Lambert-Bouguer Law -- 1.4 Effect of the Environment on Absorption Spectra -- References -- 2 Determination of the CalcofluorWhite Molar Extinction Coefficient Value in the Absence and Presence of a1-Acid Glycoprotein -- 2.1 Introduction -- 2.2 Biological Material Used -- 2.2.1 Calcofluor White -- 2.2.2 a1-Acid glycoprotein -- 2.3 Experiments -- 2.3.1 Absorption spectrum of Calcofluor free in PBS buffer -- 2.3.2 Determination of e. value of CalcofluorWhite free in PBS buffer -- 2.3.3 Determination of CalcofluorWhite e. value in the presence of a1-acid glycoprotein -- 2.4 Solution -- References -- 3 Determination of Kinetic Parameters of Lactate Dehydrogenase -- 3.1 Objective of the Experiment -- 3.2 Absorption Spectrum of NADH -- 3.3 Absorption Spectrum of LDH -- 3.4 Enzymatic Activity of LDH -- 3.5 Kinetic Parameters -- 3.6 Data and Results -- 3.6.1 Determination of enzyme activity -- 3.6.2 Determination of kinetic parameters -- 3.7 Introduction to Kinetics and the Michaelis-Menten Equation -- 3.7.1 Definitions -- 3.7.2 Reaction rates -- References -- 4 Hydrolysis of p-Nitrophenyl-B-D-Galactoside with B-Galactosidase from E. coli -- 4.1 Introduction -- 4.2Solutions to be Prepared -- 4.3 First-day Experiments -- 4.3.1 Absorption spectrum of PNP -- 4.3.2 Absorption of PNP as a function of pH -- 4.3.3 Internal calibration of PNP -- 4.3.4 Determination of B-galactosidase optimal pH -- 4.3.5 Determination of B-galactosidase optimal temperature -- 4.4 Second-day Experiments -- 4.4.1 Kinetics of p-nitrophenyl-B-D-galactoside hydrolysis with B-galactosidase -- 4.4.2 Determination of the B-galactosidase concentration in the test tube -- 4.5 Third-day Experiments.

4.5.1 Determination of Km and Vmax of B-galactosidase -- 4.5.2 Inhibiton of hydrolysis kinetics of p-nitrophenyl-B-D-galactoside -- 4.6 Fourth-day Experiments -- 4.6.1 Effect of guanidine chloride concentration on B-galactosidase activity -- 4.6.2 OD variation with guanidine chloride -- 4.6.3 Mathematical derivation of Keq -- 4.6.4 Definition of the standard Gibbs free energy, G. -- 4.6.5 Relation between G. and G -- 4.6.6 Relation between G. and Keq -- 4.6.7 Effect of guanidine chloride on hydrolysis kinetics of p-nitrophenyl-B-D-galactoside -- References -- 5 Starch Hydrolysis by Amylase -- 5.1 Objectives -- 5.2 Introduction -- 5.3 Materials -- 5.4Procedures and Experiments -- 5.4.1 Preparation of a 20 g lŠ1starch solution -- 5.4.2 Calibration curve for starch concentration -- 5.4.3 Calibration curve for sugar concentration -- 5.4.4 Effect of pH -- 5.4.5 Temperature effect -- 5.4.6 Effect of heat treatment at 90.C -- 5.4.7 Kinetics of starch hydrolysis -- 5.4.8 Effect of inhibitor (CuCl2) on the amylase activity -- 5.4.9Effect of amylase concentration -- 5.4.10 Complement experiments that can be performed -- 5.4.11 Notes -- References -- 6 Determination of the pK of a Dye -- 6.1 Definition of pK -- 6.2 Spectrophotometric Determination of pK -- 6.3 Determination of the pK of 4-Methyl-2-Nitrophenol -- 6.3.1 Experimental procedure -- 6.3.2 Solution -- Reference -- 7 Fluorescence Spectroscopy Principles -- 7.1 Jablonski Diagram or Diagram of Electronic Transitions -- 7.2 Fluorescence Spectral Properties -- 7.2.1 General features -- 7.2.2 Stokes shift -- 7.2.3 Relationship between the emission spectrum and excitation wavelength -- 7.2.4 Inner filter effect -- 7.2.5 Fluorescence excitation spectrum -- 7.2.6 Mirror-image rule -- 7.2.7 Fluorescence lifetime -- 7.2.8 Fluorescence quantum yield -- 7.2.9 Fluorescence and light diffusion.

7.3 Fluorophore Structures and Properties -- 7.3.1 Aromatic amino acids -- 7.3.2 Cofactors -- 7.3.3 Extrinsinc fluorophores -- 7.4 Polarity and Viscosity Effect on Quantum Yield and Emission Maximum Position -- References -- 8 Effect of the Structure and the Environment of a Fluorophore on Its Absorption and Fluorescence Spectra -- Experiments -- Questions -- Answers -- Reference -- 9 Fluorophore Characterization and Importance in Biology -- 9.1 Experiment 1. Quantitative Determination of Tryptophan in Proteins in 6 M Guanidine -- 9.1.1 Introduction -- 9.1.2 Principle -- 9.1.3 Experiment -- 9.1.4 Results obtained with cytochrome b2 core -- 9.2 Experiment 2. Effect of the Inner Filter Effect on Fluorescence Data -- 9.2.1 Objective of the experiment -- 9.2.2 Experiment -- 9.2.3 Results -- 9.3 Experiment 3. Theoretical Spectral Resolution of Two Emitting Fluorophores Within a Mixture -- 9.3.1 Objective of the experiment -- 9.3.2 Results -- 9.4 Experiment 4. Determination of Melting Temperature of Triglycerides in Skimmed Milk Using Vitamin A Fluorescence -- 9.4.1 Introduction -- 9.4.2 Experiment to conduct -- 9.4.3 Results -- References -- 10 Fluorescence Quenching -- 10.1 Introduction -- 10.2 Collisional Quenching: the Ster-Volmer Relation -- 10.3Different Types of Dynamic Quenching -- 10.4 Static Quenching -- 10.4.1 Theory -- 10.5 Thermal Intensity Quenching -- References -- 11 Fluorescence Polarization -- 11.1 Definition -- 11.2 Fluorescence Depolarization -- 11.2.1 Principles and applications -- 11.3 Fluorescence Anisotropy Decay Time -- 11.4 Depolarization and Energy Transfer -- References -- 12 Interaction Between Ethidium Bromide and DNA -- 12.1 Objective of the Experiment -- 12.2 DNA Extraction from Calf Thymus or Herring Sperm -- 12.2.1 Destruction of cellular structure -- 12.2.2 DNA extraction -- 12.2.3 DNA purification.

12.2.4 Absorption spectrum of DNA -- 12.3 Ethidium Bromide Titration with Herring DNA -- 12.4 Results Obtained with Herring DNA -- 12.4.1 Absorption and emission spectra -- 12.4.2 Analysis and interpretation of the results -- 12.5 Polarization Measurements -- 12.6 Results Obtained with Calf Thymus DNA -- 12.7 Temperature Effect on Fluorescence of the Ethidium Bromide-DNA Complex -- References -- 13 Lens culinaris Agglutinin: Dynamics and Binding Studies -- 13.1 Experiment 1. Studies on the Accessibility of IŠ to a Fluorophore: Quenching of Fluorescein Fluorescence with KI -- 13.1.1 Objective of the experiment -- 13.1.2 Experiment -- 13.1.3 Results -- 13.2 Experiment 2. Measurement of Rotational Correlation Time of Fluorescein Bound to LCA with Polarization Studies -- 13.2.1 Objective of the work -- 13.2.2 Polarization studies as a function of temperature -- 13.2.3 Polarization studies as a function of sucrose at 20.C -- 13.2.4 Results -- 13.3 Experiment 3. Role of a-L-fucose in the Stability of Lectin-Glycoproteins Complexes -- 13.3.1 Introduction -- 13.3.2 Binding studies -- 13.3.3 Results -- References -- 14 Förster Energy Transfer -- 14.1 Principles and Applications -- 14.2 Energy-transfer Parameters -- 14.3 Bioluminescence Resonance Energy Transfer -- References -- 15 Binding of TNS on Bovine Serum Albumin at pH 3 and pH 7 -- 15.1 Objectives -- 15.2Experiments -- 15.2.1 Fluorescence emission spectra of TNS-BSA at pH 3 and 7 -- 15.2.2 Titration of BSA with TNS at pH 3 and 7 -- 15.2.3 Measurement of energy-transfer efficiency fromTrp residues to TNS -- 15.2.4 Interaction between free Trp in solution and TNS -- 15.3 Results -- 16 Comet Test for Environmental Genotoxicity Evaluation: A Fluorescence Microscopy Application -- 16.1 Principle of the Comet Test -- 16.2 DNA Structure -- 16.3 DNA Reparation -- 16.4 Polycyclic Aromatic Hydrocarbons.

16.5 Reactive Oxygen Species -- 16.6 Causes of DNA Damage and Biological Consequences -- 16.7 Types of DNA Lesions -- 16.7.1 Induction of abasic sites, AP, apurinic, or apyrimidinic -- 16.7.2 Base modification -- 16.7.3 DNA adducts -- 16.7.4 Simple and double-stranded breaks -- 16.8 Principle of Fluorescence Microscopy -- 16.9 Comet Test -- 16.9.1 Experimental protocol -- 16.9.2 Nature of damage revealed with the Comet test -- 16.9.3 Advantages and limits of the method -- 16.9.4 Result expression -- References -- 17 Questions and Exercises -- 17.1 Questions -- 17.1.1 Questions with short answers -- 17.1.2 Find the error -- 17.1.3 Explain -- 17.1.4 Exercises -- 17.2 Solutions -- 17.2.1 Questions with short answers -- 17.2.2 Find the error -- 17.2.3 Explain -- 17.2.4 Exercises solutions -- Index -- Color plate.