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.

Auteur

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

Contenu
1 Absorption Spectroscopy Theory 1

1.1 Introduction 1

1.2 Characteristics of an Absorption Spectrum 2

1.3 BeerLambertBouguer 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 1-Acid Glycoprotein 13

2.1 Introduction 13

2.2 Biological Material Used 13

2.2.1 Calcofluor White 13

2.2.2 **1-Acid glycoprotein 13

2.3 Experiments 16

2.3.1 Absorption spectrum of Calcofluor free in PBS buffer 16

2.3.2 Determination of . value of Calcofluor White free in PBS buffer 16

2.3.3 Determination of Calcofluor White . value in the presence of 1-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 MichaelisMenten Equation 26

3.7.1 Definitions 26

3.7.2 Reaction rates 26

References 32

4 Hydrolysis of p-Nitrophenyl--D-Galactoside with -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 -galactosidase optimal pH 39

4.3.5 Determination of -galactosidase optimal temperature 40

4.4 Second-day Experiments 40

4.4.1 Kinetics of p-nitrophenyl--D-galactoside hydrolysis with -galactosidase 40

4.4.2 Determination of the -galactosidase concentration in the test tube 42

4.5 Third-day Experiments 44

4.5.1 Determination of Km and Vmax of -galactosidase 44

4.5.2 Inhibiton of hydrolysis kinetics of p-nitrophenyl--D-galactoside 45

4.6 Fourth-day Experiments 47

4.6.1 Effect of guanidine chloride concentration on -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, G' 51

4.6.5 Relation between G' and G' 51

4.6.6 Relation between G' and *K*eq 52

4.6.7 Effect of guanidine chloride on hydrolysis kinetics of p-nitrophenyl--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 l1starch 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 90C 69

5.4.7 Kinetics of starch hydrolysis 70 5.4.8 Effect of inhibitor (CuCl2) on the amyla...