Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies (eBook)
426 Seiten
Elsevier Science (Verlag)
978-0-08-047448-9 (ISBN)
* Offers concise information on the fundamentals of absorption and fluorescence
* Critically reviews examples taken from previously published literature
* Highly illustrated, it is suitable for academic and institutional libraries and government laboratories
Structure and Dynamics of Macromolecules: Absorption and Fluorescence Studies is clearly written and contains invaluable examples, coupled with illustrations that demonstrate a comprehensible analysis and presentation of the data. This book offers practical information on the fundamentals of absorption and fluorescence, showing that it is possible to interpret the same result in different ways. It is an asset to students, professors and researchers wishing to discover or use absorption and fluorescence spectroscopy, and to scientists working on the structure and dynamics of macromolecules.* Offers concise information on the fundamentals of absorption and fluorescence * Critically reviews examples taken from previously published literature * Highly illustrated, it is suitable for academic and institutional libraries and government laboratories
Cover 1
Preface 5
Contents 7
Light Absorption by a Molecule 13
Jablonski Diagram or Diagram of Electronic Transitions 13
Singlet and Triplet States 14
Forbidden and Non Forbidden Transitions 19
Reading the Jablonski Diagram 19
Chemical Bonds 20
Atomic and Molecular Orbitals 20
The Coordinated Bond 21
Absorption Spectroscopy 23
Origin and Properties of the Absorption Spectra 23
Beer-Lambert-Bouguer Law 26
Determination of the Molar Extinction Coefficient of Proteins 30
Effect of High Optical Densities on the Beer-Lambert-Bouguer Law 33
Effect of the Environment on the Absorption Spectra 33
Absorption Spectroscopy and Electron Transfer Mechanism in Proteins 39
Second - Derivative Absorption Spectroscopy 52
Theory 52
Binding of Progesterone to Alpha1-Acid Glycoprotein 59
Fluorescence: Principles and Observables 67
Introduction 67
Fluorescence Properties 70
Stokes Shift 70
Relation between Emission Spectrum and Excitation Wavelength 72
Relation between the Fluorescence Intensity and the Optical Density 74
Fluorescence Excitation Spectrum 78
The Mirror-Image Rule 81
Fluorescence Lifetime 82
Definition of the Fluorescence Lifetime 82
Mean Fluorescence Lifetime 84
Fluorescence Lifetime Measurement 85
Time Correlated Single Photon Counting 91
The Strobe Technique 96
Excitation with Continuous Light: the Phase and Demodulation Method 97
Principles 97
Multifrequency and Cross-Correlation 100
Fluorescence Quantum Yield 103
Fluorescence and Light Diffusion 110
Fluorophores: Descriptions and Properties 111
Introduction 111
Types of Fluorophores 111
Intrinsic Fluorophores 111
Aromatic Amino Acids 111
The Co-factors 123
Extrinsinc Fluorophores 125
Fluorescein and Rhodamine 125
Naphthalene Sulfonate 126
Nucleic Bases 135
Ions Detectors 136
Carbohydrates Fluorescent Probes 139
Oxidation of Tryptophan Residues with N-bromosuccinimide 141
Nitration of Tyrosine Residues with Tetranitromethane (TNM) 143
Effect of the Environment on the Fluorescence Observables 146
Polarity Effect on the Quantum Yield and the Position of the Emission Maximum 146
Effect of the Viscosity on the Fluorescence Emission Spectrum 148
Effect of the Environment on the Fluorescence Lifetime 151
Relation between Fluorescence and a Specific Sequence in a Protein 152
Fluorescence Quenching 153
Introduction 153
Collisional Quenching : the Stern-Volmer Relation 153
The Different Types of Dynamic Quenching 157
Static Quenching 170
Theory 170
Cytochrome c - Cytochrome b2 Core Interaction 173
Cytochrome b2 Core - Flavodehydrogenase Interaction 174
Determination of Drug Binding to Alpha1 - Acid Glycoprotein 176
Comparison between Dynamics and Static Quenching 178
Combination of Dynamic and Static Quenching 181
Thermal Intensity Quenching 183
Photoquenching 203
Fluorescence Polarization 205
Aims and Definition 205
Principles of Polarization or of Photoselection 206
Absorption Transitions and Excitation Polarization Spectrum 209
Fluorescence Depolarization 211
Principles and Applications 211
Measurements of Rotational Correlation Time of Tyrosine in Small Peptides 218
Fluorescence Lifetime 218
Fluorescence Intensity Quenching of Tyrosine Residues by Iodide 218
Quenching Emission Anisotropy 220
DNA-Protein Interaction 221
Fluorescence Anisotropy Decay Time 223
Depolarization and Energy Transfer 226
Forster Energy Transfer 229
Principles 229
Energy Transfer Parameters 236
Relation between Energy Transfer and Static Quenching 239
Origin of Proteins Fluorescence 249
Description of the Structure and Dynamics of Alpha1-Acid Glycoprotein by Fluorescence Studies 273
Introduction 273
Methods 278
Fluorescence Properties of TNS Bound to Sialylated Alpha1-Acid Glycoprotein 279
Binding Parameters 279
Fluorescence Lifetime 282
Dynamics of TNS Bound to Alpha1-Acid Glycoprotein 282
Fluorescence Properties of Calcofluor Bound to Alpha-Acid Glycoprotein 286
Fluorescence Parameters of Calcofluor Bound to Alpha1-Acid Glycoprotein 286
Binding Parameters 288
Nature of the Interaction of Calcofluor with Alpha1 -Acid Glycoprotein and HSA 289
Titration of Carbohydrate Residues with Calcofluor 292
Fluorescence Lifetime of Calcofluor Bound to Alpha1-Acid Glycoprotein 297
Binding Parameters of Calcofluor White - Alpha1-Acid Glycoprotein by following the Fluorescence Lifetime of Calcofluor 299
Dynamics of Calcofluor Bound to Alpha1-Acid Glycoprotein at Equimolar Concentrations 300
Dynamics of Calcofluor Bound to Sialylated Alpha1-Acid Glycoprotein 300
Dynamics of Calcofluor Bound to Asialylated Alpha1 - Acid Glycoprotein 301
Dynamics of Calcofluor Bound to Alpha1-Acid Glycoprotein at Excess Concentration of Calcofluor 303
Fluorescence Properties of the Trp Residues in Alpha1-Acid Glycoprotein 305
Fluorescence Spectral Properties 305
Effect of High Calcofluor Concentration on the Local Structure of Alpha1-Acid Glycoprotein 308
Deconvolution of the Emission Spectra Obtained at Low and High Concentrations of Calcofluor into Different Components 309
Analysis as a Sum of Gaussian Bands 309
The Ln-Normal Analysis 311
Forster Energy Transfer Experiments from Trp Residues to Calcofluor White 317
Relation between the Secondary Structure of Carbohydrate Residues of Alpha1-Acid Glycoprotein and the Fluorescence of the Trp Residues of the Protein 321
Effect of the Secondary Structure of Carbohydrate Residues of Alpha1-Acid Glycoprotein on the Local Dynamics of the Protein 325
Fluorescence Emission Intensity of Alpha1-Acid Glycoprotein as a Function of Temperature 325
Fluorescence Emission Anisotropy of Alpha1-Acid Glycoprotein as a Function of Temperature 327
Tertiary Structure of Alpha1-Acid Glycoprotein : First Model Describing the Presence of a Pocket 331
Are there any other Alternative Fluorescence Methods other than the QREA or the Weber's Method to Put into Evidence the Presence of a Pocket within Alpha1-Acid Glycoprotein 335
Experiments giving Proofs of the Presence of a Pocket within Alpha1-Acid Glycoprotein 336
Binding of Progesterone to Alpha1-Acid Glycoprotein 336
Binding of Hemin to Alpha1- Acid Glycoprotein 339
Homology Modeling of Alpha1- Acid Glycoprotein 339
Dynamics of Trp Residues in Crystals of Human Alpha1- Acid Glycoprotein 344
Introduction 344
Protein Preparation 344
Fluorescence Excitation and Emission Spectra 346
Dynamics of the Microenvironments of the Hydrophobic Trp Residues 348
Structural Studies of Human Alpha1-Acid Glycoprotein followed by X-Rays Scattering and Transmission Electron Microscopy 351
Small Angle Diffraction Studies (SAXS) 351
Wide Angle Diffraction Studies (WAXS) 353
Carbohydrate Residues Studies 353
Electron Microscopy Studies 354
Structure and Dynamics of Hemoglobin Subunits and of Myoglobin 357
Introduction 357
Dynamics of Trp Residues in Hemoglobin and in its Subunits 358
Properties of Protoporphyrin IX in Different Solvents and in Apomyoglobin 361
Chemical Structure of Porphyrins 361
Spectral Properties of Protoporphyrin IX in Different Solvents 363
Spectral Properties of Protoporphyrin IX Bound to Apomyoglobin (MbdesFe) 369
Dynamics of Protoporphyrin IX Embedded in the Heme Pocket 371
Protein Rotational Correlation Time 371
Activation Energy of the Porphyrin Motions in the Heme-Pocket 371
Residual Internal Motions of Porphyrin 372
Effect of Metal on the Porphyrin Dynamics 374
Dynamics of the Protein Matrix and the Heme Pocket 376
Significance of the Upward Curvature 379
Effect of Sucrose on the Bimolecular Diffusion Constant 382
Fluorescence Fingerprints of Animal and Vegetal Species and/or Varieties 385
Fluorescence Fingerprints of Eisenia Fetida and Eisenia Andrei 385
Introduction 385
Results 386
Structural Characterization of Varieties of Crops among a Species and of Genetically Modified Organisms: A Fluorescence Study 389
Pioneering Work of Zandomeneghi 389
Structural Characterization of Crops 391
References 399
Index 421
Light Absorption By A Molecule
1 Jablonski diagram or diagram of electronic transitions
Absorption of light (photons) by a population of molecules induces the passage of electrons from the single ground electronic level So to an excited state Sn (n > 1). In the excited state, a molecule is energetically unstable and thus it should return to the ground state So. This will be achieved according to two successive steps:
- The molecule at the excited state Sn will dissipate a part of its energy in the surrounding environment reaching by that the lowest excited state S1.
- From the excited state S1, the molecule will attain the ground state So via different competitive processes:
a) Emission of a photon with a radiative rate constant kr. Emission of a photon is called fluorescence.
b) The energy absorbed by the molecules is dissipated in the medium as heat. This type of energy is non radiative and occurs with a rate constant ki.
c) The excited molecules can give up their energy to molecules located near by. This energy transfer occurs with a rate constant kq (collisional quenching), or with a rate constant kt (energy transfer at distance).
d) A transient passage occurs to the excited triplet state T1 of energy lower than S1 with a rate constant kisc. The triplet state is energetically unstable. Therefore, deexcitation of the molecule will occur via different competitive phenomena:
- Emission of a photon with a rate constant kp. This phenomenon is called phosphorescence.
- Dissipation of non radiative energy with a rate constant k'i.
- Transfer of energy to another molecule at distance (rate constant k't) or by collision (rate constant k’q) (Figure 1.1).
The S → S or the T → T transitions are called internal conversion.
The S1 -→ T1 transition is called intersystem crossing. For each excited state S, an excited state T does exist.
Molecules that absorb photons are called chromophores and a chromophore that emits a photon is called a fluorophore. Therefore, a fluorophore is inevitably a chromophore, however a chromophore is not necessarily a fluorophore. For example, heme does absorb light however it does not fluoresce. The absence of fluorescence is the result of total energy transfer from the porphyrin ring to the iron. Other metals such as zinc or tin do not quench completely porphyrin fluorescence. Thus, zincporphyrin and tinporphyrin display fluorescence of energy lower than that of porphyrin.
It is important to indicate that thermal activation of a molecule can induce its passage from the ground to the excited state.
Used with permission. ©Thomas G. Chasteen, Sam Houston State University
2 Singlet and triplet states
Transitions described in Jablonski diagram take place within chemical molecules and therefore they concern the atoms that constitute these molecules. The electrons of these molecules are responsible for the different transitions shown in the Jablonski diagram. For this reason, the Jablonski diagram is also called the electronic transitions diagram.
Since localization of an electron is difficult, one referred to four quantum numbers (n, 1, m and s) to characterize an electron and to differentiate it from the others. The principal quantum number n determines the energy of any one-electron atom of nuclear charge Z. n can assume any positive integral value, excluding zero.
The angular – momentum quantum number 1 determines the angular momentum of the electron. It may assume all integral values from 0 to n-1 inclusive.
The magnetic quantum number m characterizes the magnetic field generated by the electric current of the electron, circulating in a loop, m can assume all integral values between −1 and + 1 including zero.
The spin quantum number s is the result of the electron spinning about its own axis. Thus, a local small magnet is generated with a spin s.
Singlet and triplet states depend on the quantum number of spin of the electron (s). According to Pauli Exclusion Principle, two electrons within a defined orbital cannot have equal four quantum numbers and thus will differ by the spin number. Two spin numbers are attributed to an electron, + ½ and – ½. Therefore, two electrons that belong to the same orbital will have opposite spin.
A parameter called the Multiplicity (M) is defined as being equal to:
=s1+s2+1
(1.1)
When the spins are parallel, M will be equal to 1 and we shall have the singlet state S.
When the spins are anti-parallel, M will be equal to 3. We are in presence of a triplet state T (Figure 1.2).
Upon excitation, one electron absorbs light energy and goes to an upper vacant orbital. The molecule is in the excited state Sn. After desexcitation, the electron will return to its original orbital and the molecule will be in the ground state So.
In the ground state, all molecules except oxygen are in the singlet form. Oxygen is in the triplet state, and when excited it reaches the singlet state. The singlet excited state of oxygen is destructive for cells attained by tumors. Photodynamic therapy is a process in which a light-responsive chemical, when exposed to the appropriate wavelength of light, is activated to undergo either a photophysical process or to initiate photochemistry, producing molecular species which can interact with biological targets (photosensitization).
Such interactions can be exploited for biomedical applications or for basic studies. Photosensitization is exploited for the destruction of tumors and certain non-neoplastic target tissues in an approach termed photodynamic therapy (PDT). Compounds such as porphyrins are localized in target cells and tissues. Upon light activation, energy transfer occurs from porphyrins to oxygen molecules inducing by that a triplet - > singlet transition within the oxygen state. Cells attained by tumor attract excited oxygen molecules and then are being destroyed by these same oxygen molecules. The history of photodetection and photodynamic therapy has been described by Ackroyd et al. (2001). Administrated drugs can also be used instead of porphyrins to induce singlet oxygen formation in cells.
Perotti et al (2004) studied the efficacy of 5-aminolevulinic acid (ALA) derivatives as pro-photosensitising agent. The authors used cell line LM3 for their studies. ALA is a precursor of protoporphyrin IX and thus injection of ALA and / or of its derivatives into cells will induce the formation of protoporphyrin IX, a necessary molecule to induce singlet oxygen and thus cell death.
Figure 1.3 describes porphirin synthesis from ALA and its derivatives. One can notice that synthesis of porphyrin does not follow the same rule for all the coupounds used.
Figure 1.4 displays dark and PDT toxicities for ALA and its derivative. Undecanoyl-ALA is intrinsically toxic to cells even in the absence of light (Panel A). Increasing light doses accelerates cell death (Panels B and C).
Finally, the authors calculated tumor porphyrin synthesis after administration of equimolar concentrations of ALA and ALA derivatives to mice. They found that ALA is the best inducer of porhyrin synthesis (15.9 ± 1.12 nmol/g of tissue), followed by THP-ALA (8.83 ± 0.95 nmol/g of tissue), He-ALA (3.11 ± 0.42 nmol/g of tissue) and undecanoyl-ALA (2.64 ± 0.23 nmol/g of tissue). The values were calculated after three hours of ALA or ALA derivative administration to the cell lines.
The natural non-toxic hypericin is a photosensitizing anti-cancer drug. In fact, neoplastic cells in culture respond to hypericin in a dose-dependent fashion: high doses of light and high...
| Erscheint lt. Verlag | 30.8.2011 |
|---|---|
| Sprache | englisch |
| Themenwelt | Sachbuch/Ratgeber |
| Naturwissenschaften ► Chemie ► Analytische Chemie | |
| Naturwissenschaften ► Chemie ► Physikalische Chemie | |
| Naturwissenschaften ► Chemie ► Technische Chemie | |
| Naturwissenschaften ► Geowissenschaften ► Mineralogie / Paläontologie | |
| Naturwissenschaften ► Physik / Astronomie | |
| Technik ► Umwelttechnik / Biotechnologie | |
| Wirtschaft | |
| ISBN-10 | 0-08-047448-9 / 0080474489 |
| ISBN-13 | 978-0-08-047448-9 / 9780080474489 |
| Haben Sie eine Frage zum Produkt? |
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