Spectrophotometry and Spectrofluorimetry A Practical Approach

by Gore, Michael G.

• ISBN13: 9780199638123
• ISBN10: 0199638128
• Edition: 2nd
• Format: Paperback
• Copyright: 2000-06-08
• Publisher: Oxford University Press

Summary

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.

Table of Contents

List of protocols	xv
Abbreviations	xvii
Introduction to light absorption: visible and ultraviolet spectra	1	(32)
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 occurring chromophores	6	(3)
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' spectrophotometers	9	(1)
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 conditions	17	(5)
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 cytochrome difference spectrum (reduced minus oxidized)	27	(3)
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 processes	35	(4)
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 phosphorescence spectroscopy	69	(30)
Thomas D. Bradrick
Jorge E. Churchich
Introduction	69	(1)
Background	69	(15)
Basic photophysics and time dependence of fluorescence and phosphorescence decays	69	(2)
Fluorescence and phosphorescence energy transfer and sensitized luminescence	71	(1)
Observed time dependence of fluorescence	72	(3)
Decay associated spectra (DAS) and discrete lifetimes versus lifetime distributions	75	(1)
Polarized excitation and emission anisotropy decay	75	(3)
Data analysis	78	(6)
Equipment for time-resolved fluorescence measurements	84	(5)
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 transition metal complexes	117	(2)
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 structural units in a protein from peptide region CD spectra	125	(3)
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 binding isotherms for multiple ligand-macromolecule interactions using spectroscopic methods	141	(26)
Wlodzimierz Bujalowski
Maria J. Jezewska
Introduction	141	(2)
Thermodynamic basis of quantitative spectroscopic titrations	143	(20)
The signal used to monitor ligand-macromolecule interactions originates from the macromolecule	144	(10)
Signal used to monitor the interactions originates from the ligand	154	(9)
Summary	163	(4)
Acknowledgement	164	(1)
References	164	(3)
Steady-state kinetics	167	(16)
Athel Cornish-Bowden
Introduction to rate equations, first-order, second-order reactions etc.	167	(1)
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 aspects of, assay design	185	(2)
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 of two methods	197	(2)
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 acid side chains	204	(2)
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 experiment	212	(1)
The form of a simple progress curve: Making sure the apparatus is mixing and transferring reactants to the observation chamber rapidly	213	(1)
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 `rapid scan methods'	227	(14)
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 dead time	243	(1)
Measuring mixing efficiency	243	(1)
Sample preparation	244	(1)
Artefacts	245	(1)
Temperature effects	245	(1)
Density differences between the two solutions	246	(1)
Factors affecting the sensitivity of the optical system	246	(3)
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 fluorescence to follow binding reactions	250	(7)
Use of ligand fluorescence to monitor binding reactions	257	(3)
Extrinsic probes	260	(2)
Examples of reactions monitored by changes in fluorescence of covalently attached fluorophores	262	(1)
References	263	(2)
Stopped-flow circular dichroism	265	(18)
Alison Rodger
Michael J. Carey
Introduction	265	(1)
Instrumentation considerations	266	(5)
Available budget	266	(1)
Sensitivity required	267	(1)
Minimum dead time required	267	(1)
Corrosiveness and adsorbance of the samples to be studied	267	(1)
Type of flow system: stepper motor or compressed air driven	268	(1)
Size and design of the optical cell	268	(1)
The number of syringes and mixing stages required	269	(1)
Mixing ratios required and whether these need to be variable from experiment to experiment	269	(1)
Sample viscosity	269	(1)
Wavelength range for detection, wavelength scanning and bandwidth	270	(1)
Software	270	(1)
Currently available instrumentation	271	(3)
Stopped-flow attachment for CD spectropolarimeters	271	(2)
Integrated stopped-flow CD systems	273	(1)
Additional experimental considerations	274	(4)
Parameters	274	(1)
Zero-time and dead-time calibration	275	(1)
Baseline	275	(1)
System tests	275	(3)
Examples	278	(5)
Stopped-flow CD and lysozyme folding	279	(1)
DNA as a catalytic template	280	(1)
References	281	(2)
Spectrophotometry and fluorimetry of cellular compartments and intracellular processes	283	(24)
C. Lindsay Bashford
Introduction	283	(1)
Experimental design	284	(9)
Apparatus	284	(3)
Light sources	287	(1)
Wavelength selection	288	(1)
Chromophore selection	288	(4)
Characterization and calibration of optical signals	292	(1)
Examples	293	(11)
Oxidation-reduction state of tissue mitochondria	293	(2)
Membrane potential of cells and organelles	295	(4)
pH of cellular compartments	299	(4)
Membrane cycling and recycling	303	(1)
Future prospects	304	(3)
Acknowledgements	304	(1)
References	304	(3)
Use of optical spectroscopic methods to study the thermodynamic stability of proteins	307	(22)
Maurice R. Eftink
Haripada Maity
Introduction	307	(1)
Basic thermodynamic principles	308	(8)
The two-state model	308	(1)
Thermal unfolding	309	(1)
Denaturant induced unfolding	309	(1)
Acid induced unfolding	310	(1)
Pressure induced unfolding	310	(1)
Simulations	311	(1)
Unfolding of oligomeric proteins	311	(5)
Practical considerations and deviations from the two-state model	316	(4)
Existence of equilibrium intermediates	316	(1)
Kinetic considerations	316	(1)
Irreversibility	317	(1)
Baseline considerations	318	(1)
Interfering substances	319	(1)
Global analysis	320	(1)
Advantages of different spectroscopic signals	320	(5)
Absorbance	320	(2)
Circular dichroism	322	(1)
Fluorescence	323	(2)
Concluding remarks	325	(4)
Acknowledgements	325	(1)
References	326	(3)
The use of spectroscopic techniques in the study of DNA stability	329	(28)
John SantaLucia Jr
Introduction	329	(1)
Overview of UV melting	330	(4)
Strengths and weaknesses of UV melting and calorimetry	333	(1)
Sample	334	(9)
Sequence design	334	(1)
Redundant design of motifs	335	(1)
Sample preparation	336	(3)
Choice of buffer	339	(4)
Instrumentation	343	(5)
Microvolume cuvettes and aluminium cuvette adapters	343	(1)
Spectrophotometer	344	(4)
Data analysis	348	(9)
Curve fitting to calculate thermodynamic parameters	348	(4)
Presentation of normalized absorbance curves	352	(1)
Error analysis	353	(1)
References	354	(3)
A1 List of suppliers	357	(6)
Index	363