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List of protocols xv
Abbreviations xvii
Introduction to light absorption: visible and 1 (32)
ultraviolet spectra
Robert K. Poole
Uldis Kalnenieks
Introduction 1 (1)
Radiation and light 1 (1)
UV and visible spectra 2 (1)
Spectrophotometry 2 (4)
The Beer-Lambert law 2 (1)
Deviations from the Beer-Lambert law 3 (1)
Absorbance or light scattering? 4 (2)
Spectra of some important naturally 6 (3)
occurring chromophores
Amino acids and proteins 6 (1)
Nucleic acids 6 (1)
NAD(P)H 7 (1)
Carotenoids 7 (1)
Haem proteins 7 (2)
Spectrophotometer configurations 9 (8)
Single beam spectrophotometers 9 (1)
Split beam or `double beam' 9 (1)
spectrophotometers
Dual-wavelength spectrophotometers 10 (3)
Multi-wavelength spectrophotometers 13 (1)
Diode array spectrophotometers 13 (3)
Microwell plate-reading spectrophotometers 16 (1)
Reflectance methods 16 (1)
Novel double monochromator methods 16 (1)
Computing and spectrophotometry 17 (1)
Choice of spectrophotometer operating 17 (5)
conditions
Wavelength range and light source 17 (1)
Spectral versus natural bandwidth 17 (2)
Spectral resolution 19 (1)
Scan speed and instrument response time 20 (1)
Temperature 20 (2)
Use of the spectrophotometer 22 (11)
The choices 22 (1)
Baselines 22 (2)
Isosbestic points 24 (1)
Wavelength and absorbance calibrations 24 (1)
Choice and use of cuvettes (cells) 25 (2)
A detailed example: recording of a 27 (3)
cytochrome difference spectrum (reduced
minus oxidized)
Post-scan options 30 (1)
Acknowledgements 31 (1)
References 31 (2)
Fluorescence principles and measurement 33 (36)
Arthur G. Szabo
Introduction 33 (1)
Physical principles 33 (6)
The absorption process 33 (2)
Excited singlet state deactivation 35 (4)
processes
Fluorescence parameters 39 (1)
Fluorescence spectrum 39 (1)
Fluorescence quantum yield, FF 39 (1)
Singlet and radiative lifetime 40 (1)
Fluorescence spectrometers 40 (7)
The light source 42 (1)
Wavelength selectors 42 (2)
Sample excitation components 44 (1)
Sample compartment 44 (1)
Fluorescence path optical components 45 (1)
Fluorescence instrumentation electronics 46 (1)
Fluorescence spectra 47 (11)
Inner filter effect 47 (1)
Light scattering 48 (2)
Instrumental settings 50 (1)
Fluorescence spectral corrections 51 (3)
Excitation spectra 54 (1)
Quantum yield measurement 55 (3)
Fluorescence applications 58 (8)
Fluorescence resonance energy transfer 60 (1)
Fluorescence anisotropy 60 (1)
Protein fluorescence 61 (1)
Fluorescence quenching 62 (4)
Conclusion 66 (3)
References 66 (3)
Time-resolved fluorescence and 69 (30)
phosphorescence spectroscopy
Thomas D. Bradrick
Jorge E. Churchich
Introduction 69 (1)
Background 69 (15)
Basic photophysics and time dependence of 69 (2)
fluorescence and phosphorescence decays
Fluorescence and phosphorescence energy 71 (1)
transfer and sensitized luminescence
Observed time dependence of fluorescence 72 (3)
Decay associated spectra (DAS) and 75 (1)
discrete lifetimes versus lifetime
distributions
Polarized excitation and emission 75 (3)
anisotropy decay
Data analysis 78 (6)
Equipment for time-resolved fluorescence 84 (5)
measurements
Excitation sources 85 (1)
Detectors 86 (1)
Recording electronics 86 (3)
Phosphorescence 89 (10)
Phosphorescence of proteins 89 (4)
Time-dependent phosphorescence anisotropy 93 (2)
Acknowledgements 95 (1)
References 95 (4)
Introduction to circular dichroism 99 (42)
Alison Rodger
Matthew A. Ismail
Introduction 99 (2)
Circular dichroism 99 (2)
Optical rotatary dispersion 101 (1)
Chapter outline 101 (1)
Measuring a CD spectrum 101 (9)
The instrumentation 101 (2)
The sample 103 (1)
The cuvette 103 (2)
The baseline and zeroing 105 (1)
The parameters 105 (2)
Noise reduction 107 (3)
Equations of CD spectroscopy 110 (9)
Degenerate coupled-oscillator CD 111 (3)
Non-degenerate coupled-oscillator CD 114 (1)
Carbonyl n→π* CD 115 (2)
d-d transitions of tris-chelate 117 (2)
transition metal complexes
Optical activity (optical rotation, OR) 119 (1)
Dissymmetry factor 119 (1)
Units of CD spectroscopy 119 (2)
Circular dichroism of biomolecules 121 (20)
Introduction 121 (1)
CD of polypeptides and proteins 121 (1)
Protein UV spectroscopy 121 (2)
Protein structure determination from CD 123 (2)
Determining the percentage of different 125 (3)
structural units in a protein from
peptide region CD spectra
Other applications of protein CD 128 (1)
Membrane proteins 128 (1)
DNA geometry and CD spectra 129 (2)
UV spectroscopy of the DNA bases 131 (1)
Nucleic acid CD 132 (2)
DNA/ligand interactions 134 (4)
References 138 (1)
General CD references 139 (2)
Quantitative determination of equilibrium 141 (26)
binding isotherms for multiple
ligand-macromolecule interactions using
spectroscopic methods
Wlodzimierz Bujalowski
Maria J. Jezewska
Introduction 141 (2)
Thermodynamic basis of quantitative 143 (20)
spectroscopic titrations
The signal used to monitor 144 (10)
ligand-macromolecule interactions
originates from the macromolecule
Signal used to monitor the interactions 154 (9)
originates from the ligand
Summary 163 (4)
Acknowledgement 164 (1)
References 164 (3)
Steady-state kinetics 167 (16)
Athel Cornish-Bowden
Introduction to rate equations, 167 (1)
first-order, second-order reactions etc.
Units 168 (1)
Basic assumptions in steady-state kinetics 169 (1)
Measurement of specific activity 170 (2)
Graphical determination of Km and V 172 (2)
Inhibition of enzyme activity 174 (1)
Specificity 175 (1)
Activators 176 (1)
Environmental effects on enzyme activity 177 (2)
pH 177 (1)
Temperature 178 (1)
Cooperativity 179 (2)
Experimental conditions for kinetic studies 181 (1)
Concluding remarks 182 (1)
References 182 (1)
Spectrophotometric assays 183 (26)
T.J. Mantle
D.A. Harris
Introduction 183 (2)
Spectrophotometers 183 (1)
Beer-Lambert Law 184 (1)
The nature of the sample 184 (1)
Some general comments on, and practical 185 (2)
aspects of, assay design
Accuracy and precision 186 (1)
End point and rate assays 187 (2)
End point assays 187 (1)
Rate assays 188 (1)
Rate assays involving amplification 189 (1)
Spectrophotometric assays for proteins 189 (5)
A280 190 (1)
The Biuret method 191 (1)
The Lowry method 191 (1)
The bicinchoninic assay 192 (1)
Dye-binding assay 193 (1)
Fluorimetric assay 193 (1)
Spectrophotometric assays for nucleic acids 194 (1)
Enzyme-based spectrophotometric assays 195 (4)
Some general points on assay design 195 (1)
Amount of enzyme required 195 (2)
Determination of glucose---a comparison 197 (2)
of two methods
Luminescence-based assays 199 (1)
Spectrophotometric assays of enzymes 200 (4)
Some elementary enzyme kinetics 200 (1)
Continuous assays 201 (1)
Stopped assays 201 (2)
Coupled assays 203 (1)
Plate readers 203 (1)
Centrifugal analysers 203 (1)
Spectrophotometric assays for protein amino 204 (2)
acid side chains
Cysteine 204 (1)
Lysine 205 (1)
Tyrosine 205 (1)
Histidine 206 (1)
Tryptophan 206 (1)
Concluding remarks 206 (3)
References 207 (2)
Stopped-flow spectroscopy 209 (32)
M. T. Wilson
J. Torres
Introduction 209 (1)
Features of the basic instrument 210 (2)
Instruments available 211 (1)
Measurement at a single wavelength 212 (8)
Setting up 212 (1)
Selecting the wavelength for a real 212 (1)
experiment
The form of a simple progress curve: 213 (1)
Making sure the apparatus is mixing and
transferring reactants to the observation
chamber rapidly
Measurement of the dead time 214 (3)
Amplitude of the signal 217 (1)
Assigning a signal 218 (2)
Determining rate constants 220 (7)
First-order processes 221 (2)
Second-order processes 223 (4)
Multiwavelength detection: diode array 227 (14)
`rapid scan methods'
SVD (singular value decomposition) 228 (5)
Fitting to a mechanism 233 (6)
Acknowledgements 239 (1)
References 239 (2)
Stopped-flow fluorescence spectroscopy 241 (24)
Michael G. Gore
Stephen P. Bottomley
Introduction 241 (1)
Instrumentation 241 (5)
Data collection 242 (1)
Instrument calibration, stability and 243 (1)
dead time
Measuring mixing efficiency 243 (1)
Sample preparation 244 (1)
Artefacts 245 (1)
Temperature effects 245 (1)
Density differences between the two 246 (1)
solutions
Factors affecting the sensitivity of the 246 (3)
optical system
Slit width 246 (1)
Selection of wavelength of emitted light 247 (1)
Voltage applied to PMT 247 (2)
Selection of reporter group 249 (16)
Intrinsic reporter groups 249 (1)
Examples of the use of protein 250 (7)
fluorescence to follow binding reactions
Use of ligand fluorescence to monitor

Spectrophotometry and Spectrofluorimetry: A Practical Approach Second Edition was written with the intention to help the reader understand the background concepts and practical applications of spectrophotometry and spectrofluorimetry. Optical spectroscopy underpins the day to day operationsof most laboratories in the chemical, biological and medical sciences and this edition contains substantially updated and new chapters addressing the principles of most of the more common applications such as: spectrophotometry, spectrophotometric assays, spectrofluorimetry, time resolvedfluorescence and phosphorescence studies, circular dichrosim and pre-equilibrium spectroscopic techniques. In all chapters, the emphasis is placed upon the practical aspects, with protocols to guide readers through test experiments. Other chapters are included to introduce subjects that havetraditionally depended upon spectroscopy such as basic enzyme kinetics, ligand binding, data handling and the more recently established interest in the study of protein and DNA stability. Finally, the concept of 'global analysis' is introduced to provide the reader with an insight into this methodof utilizing the vast arrays of experimental data provided by current instrumentation.