Highly Sensitive Fluorescent Methods for the Detection of Enzymes and the Determination of Their Activity by Means of Specific Hydrolases
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Sigrun Henkenjohann DISSERTATION Highly Sensitive Fluorescent Methods for the Detection of Enzymes and the Determination of their Activity by Means of Specific Hydrolases APPLIED LASER PHYSICS AND LASER SPECTROSCOPY FACULTY OF PHYSICS - BIELEFELD UNIVERSITY Printed on non-aging, wood- and acid-free paper according to DIN-ISO 9706. Gedruckt auf alterungsbeständigem, holz- und säurefreiem Papier gemäß DIN-ISO 9706. Dissertation Highly Sensitive Fluorescent Methods for the Detection of Enzymes and the Determination of their Activity by Means of Specific Hydrolases Sigrun Henkenjohann December 8th 2009 Applied Laser Physics & Laser Spectroscopy Department of Physics Bielefeld University Reviewer: Markus Sauer Andreas Hütten Zusammenfassung In sehr vielen biologischen und medizinischen Prozessen nehmen Enzyme ei- ne wichtige Schlüsselfunktion ein [1–27], auf die Einfluss genommen werden kann, wenn die betreffenden Enzyme gesteuert werden können. Dies jedoch verlangt nach einem genauen Verständnis der enzymatischen Vorgänge, wel- ches mithilfe der Fluoreszensmikroskopie verbessert werden kann. In dieser Arbeit werden zwei unterschiedliche Ansätze zur fluoreszenten Enzymologie für die Carboxypeptidase A und vier verschiedene Nukleasen vorgestellt und ausgewertet. Der erste dieser Ansätze nutzt fluoreszierende Enzymsubstrate, die sich die intrinsischen elektronspendenden Eigenschaften der natürlich vorkommenden Aminosäure Tryptophan und der Nukleinsäure Guanosin zu Nutze machen [28–34]. Verschiedene Peptidsubstrate für die Untersuchung der Carboxypeptidase A, und zwei Gruppen von sogenannten ’smart probes’ für die Untersuchung der DNaseI, DNaseII, S1-Nuklease und der DNaseX werden vorgestellt, cha- rakterisiert und im weiteren Verlauf für die Analyse spezifischer Enzymeigen- schaften angewandt. Schon kleine Unterschiede in Amino- und Nukleinsäu- resequenzen dieser Substrate führen zu großen Veränderungen der relativen Quantenausbeuten, des Hitzeverhaltens der Substrate und des Enzymumsat- zes. Die fluoreszenten Substrate werden genutzt, um den Effekt des ange- brachten Farbstoffs auf die Akzeptanz und die Umsatzgeschwindigkeit durch das Enzym zu bestimmen. In dieser Arbeit werden außerdem Untersuchun- gen zu thermischen Eigenschaften der Carboxypeptidase A, der DNaseI und der DNaseX anhand einzelner Messungen bei unterschiedlichen Temperatu- ren und durch eine innovative Methode, die sich lange stetige Phasen in der Enzymkinetik zu Nutze macht, vorgestellt. Weiterhin wird die Anwendbarkeit fluoreszenter Substrate für die Ermittlung von Michaelis-Menten Parametern und für Messungen auf Einzelmolekülebene sowie in lebenden Zellen belegt. Der zweite vorgestellte Ansatz macht von der Enzym-Immunofärbung Ge- brauch. Hier wird eine optimierte Färbemethode für die Detektion und Loka- lisierung eines spezifischen Enzyms in einer Vielzahl nativer Zelllinien ange- iii wandt. Außerdem wird eine modifizierte Nieren-Zelllinie in ihrem spezifischen Enzym-Expressionsverhalten mithilfe der konfokalen Fluoreszenzmikrosko- pie, sowie durch die hochauflösende dSTORM Methode untersucht [35]. Die vorgestellten fluoreszenten Methoden unterscheiden sich sehr stark in ihrem Wesen, ihrer Anwendbarkeit und in ihren möglichen Ergebnissen. Nichts desto trotz können sie effizient kombiniert werden und somit die Un- tersuchung einer Vielzahl biologischer und medizinischer Fragestellungen er- möglichen, was in dieser Arbeit erfolgreich präsentiert wird. Abstract Enzymes engage key roles in a wide variety of important physical and medical processes [1–27], which thus can be altered by manipulating the behavior of enzymes in charge. The capability for manipulation requires an exact under- standing of enzymatic operation modes though, which can be increased by employing fluorescence microscopy. In this work, two approaches for fluores- cence based enzyme research are presented and evaluated for Carboxypepti- dase A and four different nucleases. The first presented approach uses fluorescent enzyme substrates, which take advantage of intrinsic electron donating properties of the naturally occurring amino acid tryptophan and the nucleic acid guanosine [28–34]. Several peptide substrates for Carboxypeptidase A and two sets of smart probes for DNaseI, DNaseII, S1-Nuclease and DNaseX are introduced, char- acterized and, furthermore, utilized for the investigation of specific enzyme characteristics. Even small amino and nucleic acid sequence alterations of these substrates are found to result in strong differences concerning relative fluorescence quantum yields, thermal substrate behaviors and enzyme proces- sion velocities. With these fluorescent substrates, the effect of the attached label on enzyme acceptance and velocities is examined. Furthermore, evalua- tions of the thermal characteristics of Carboxypeptidase A, DNaseI and DNa- seX are presented by means of single measurements at various temperatures and by a novel approach, utilizing long steady state kinetics. Additionally, the suitability of fluorescent substrates for the determination of Michaelis- Menten parameters and for enzyme examinations on a single molecule scale and in living cells is proved. The presented second approach is established on the method of enzyme immunolabeling. Here, an optimized labeling method is utilized for the de- tection and localization of a specific enzyme in a variety of native cell lines. Furthermore, a modified kidney cell line is examined in its specific enzyme expression characteristics with confocal fluorescence microscopy as well as with the high resolution dSTORM method [35]. The demonstrated fluorescent approaches vary distinctly in their nature, v suitability and obtainable results. Still, they can be efficiently combined to diminish any disadvantage and allow research on a wide range of biological and medical questions, which will first be described in this work. Contents Zusammenfassung iii Abstractv 1 Introduction1 2 Theoretical Background 11 2.1 Principles of Fluorescence.................... 11 2.2 Fluorescence Spectroscopy.................... 15 2.2.1 Fluorophores....................... 15 2.2.2 Fluorescence Quenching................. 16 2.3 Fluorescence Microscopy..................... 21 2.3.1 Diffraction limited Techniques.............. 22 2.3.2 Superresolution Techniques............... 26 2.4 Enzymes.............................. 29 2.4.1 Interactions between Enzyme and Substrate...... 31 2.4.2 Enzyme Characterization................ 36 2.4.2.1 Peptidases................... 37 2.4.2.2 Nucleases.................... 39 2.5 Fluorescence based Enzyme Research.............. 41 3 Materials and Methods 47 3.1 Hardware Devices and Setups.................. 47 3.1.1 Absorption Spectrometer................ 47 3.1.2 Fluorescence Spectrophotometer............ 47 3.1.3 Multiplate-Fluorescence Reader............. 48 3.1.4 High Performance Liquid Chromatograph - HPLC.. 48 3.1.5 Zeiss Laser Scanning Microscope 710 - LSM...... 48 3.1.6 Total Internal Reflection Fluorescence Microscope.. 50 3.2 Samples and their Preparation................. 50 3.2.1 Enzymes and corresponding Buffers.......... 50 vii 3.2.2 (Fluorescent) Substrates and Modifications...... 51 3.2.3 CPA Single Molecule-Localization........... 51 3.2.4 Cell Cultures....................... 52 3.2.5 Chemicals for Immunostaining............. 55 3.2.6 Labeling Protocols.................... 56 3.3 Measurements and Data Evaluation.............. 57 3.3.1 Hairpin Computation.................. 57 3.3.2 Ensemble Measurements and Data........... 57 3.3.3 Single Molecule Measurements and Data........ 58 3.3.4 Live Cell Measurements and Data........... 59 3.3.5 LSM Measurements and Data.............. 59 3.3.6 dSTORM Measurements and Data........... 60 4 Results and Discussion 63 4.1 Enzyme Investigations with fluorescent Substrates...... 63 4.1.1 Substrate Characterization............... 64 4.1.2 Enzyme Kinetics..................... 88 4.1.3 Michaelis-Menten Kinetics................ 106 4.1.4 Determination of Label Effect to Substrate Acceptance 108 4.1.5 Evaluation of ideal Temperature Settings....... 111 4.1.6 Single Molecule Enzyme Localization.......... 117 4.1.7 Enzymes in Cells..................... 120 4.2 Enzyme Investigations via Immunostaining.......... 124 4.2.1 Labeling Methods.................... 124 4.2.2 DNaseX occurrence in native mammalian Cell Lines. 127 4.2.3 DNaseX occurrence in transfected HEK cells..... 142 4.2.4 High Resolution Measurements and Quantification of Membrane-bound DNaseX................ 147 5 Conclusion and Outlook 155 Bibliography 163 Appendix A - Publications 187 Appendix B - Protocols 191 B.1 Protocol for Preparation of Mowiol-DABCO.......... 191 B.2 Original Labeling Protocol.................... 192 B.3 Protocol for Examination of labeling Procedure........ 199 B.4 Standard labeling Protocol for triple stained mammalian Cells 206 B.5 Labeling Protocol for dSTORM Measurement......... 209 Appendix C - Additional Images 213 C.1 Mowiol®-DABCO Effect on Fluorescent Labels........ 213 C.2 Examination of labeling Procedure - Complete Set of Images 215 C.3 Complete Set of Images from dSTORM Measurement.... 217 Abbreviations KM Michaelis-Menten constant vmax maximum reaction velocity dSTORM direct Stochastically Optical Reconstruction Microscopy E. coli Escheria coli AFM Atomic Force Microscopy Antp Antennapedia AOTF Acousto-Optical Tunable Filter Ar+ Argon Ionen Asp Aspartic acid BSA Bovine Serum Albumin C-terminus Carboxyl-terminus CCD Charge-Coupled Device CPA Carboxypeptidase A CPP Cell-Penetrating Peptide DNA Deoxyribonucleic Acid ds