THE S TUDY OF APO-ENZYME/PROSTHETIC GROUPS AND THEIR APPLICATIONS IN CHEMICAL ANALYSIS by Dongxuan Shen A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in The University of Michigan 2009 Doctoral Committee: Professor Mark E. Meyerhoff, Chair Associate Professor Shuichi Takayama Associate Professor Nils G. Walter Assistant Professor Kristina I. Håkansson © Dongxuan Shen 2009 ACKNOWLEDGEMENTS First of all, I would like to thank my advisor, Dr. Mark E. Meyerhoff, for his teaching and guidance, his patience, his continuous support and encouragement throughout my dissertation work. His enthusiasm toward scientific research and his truth-seeking attitude will be a lifetime example for myself in pursuing future careers. I would also like to thank Dr. Hakansson, Dr. Takayama, and Dr. Walter for their service on my doctoral committee, especially Dr. Walter for the discussion and suggestions with synthetic PQQ work in Chapter 4. Special thanks go to my collaborators at Berry & Associates. Without their expertise in the organic synthesis, one of my PQQ projects would be impossible to advance. I want to thank specifically Dr. Jack Hodges for the preparation of PQQ derivatives and collaboration on the project described in Chapter 4. I also feel indebted to my past and present group members. I want to give special thanks to Dr. Hyoungsik Yim for his encouragement, support, and his help with starting my thesis research; Dr. Jeremy Mitchell-Koch, and Dr. Stacey Buchanan for their help during my rotation research; Dr. Sangyeul Huang for his helpful discussions with my organic synthesis work; Dr. Youngjea Kang and Dr. Wansik Cha for their insightful research idea discussions and interesting culture talks. I would also like to thank all other group members: Dr. Yiduo Wu, Dr. Zhengrong Zhou, Dr. Hairong Zhang, Dr. Fenghua ii Zhang, Dr. Jason Bennet, Jun Yang, Lin Wang, Biyun Wu, Qinyi Yan, Laura Zimmerman, Natalie Walker. You all have made my life in Michigan very enjoyable. I also appreciate all the support from my friends in JiaYin Chiristian Fellowship and AACCC: Pastor Teo, Shouchin and Michael Man, Paul and Alice Chao, Jinzhong and Yuanyuan, Xinen and Ji Fang, and all of the rest that I can’t list all of your names. Thanks for your support and prayers! Finally, I would like to thank my wife, Shiyan, my daughter, Noelle, and my mother, Okran Yang. Without their long lasting support and love, none of my work would be possible. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS..............................................................................................ii LIST OF FIGURES..........................................................................................................vi LIST OF TABLES.............................................................................................................x LIST OF ABBREVIATIONS..........................................................................................xi CHAPTER 1. INTRODUCTION 1.1. Introduction .........................................................................................................................1 1.2. Enzymes and Prosthetic Groups........................................................................................3 1.3. Enzyme-linked Homogeneous and Heterogeneous Binding Assays ...............................6 1.4. Development of a Novel Homogeneous Cleaving Enzyme Assay .................................10 1.5. Statement of Research ......................................................................................................12 1.6. References ..........................................................................................................................27 CHAPTER 2. PQQ-DOPED POLYMERIC NANOSPHERES AS SENSITIVE TRACER FOR BINDING ASSAYS 2.1. Introduction .......................................................................................................................32 2.2. Experimental......................................................................................................................34 2.3. Results and Discussion ......................................................................................................38 2.4. Conclusions ........................................................................................................................44 2.5. References ..........................................................................................................................56 CHAPTER 3. APO-GLUCOSE DEHYDROGENASE BASED HOMOGENEOUS COMPETITIVE BINDING ASSAY FOR SMALL MOLECULES USING BIOTIN/AVIDIN AS A MODEL SYSTEM .................................................................58 3.1. Introduction .......................................................................................................................58 3.2. Experimental......................................................................................................................60 3.3. Results and Discussion ......................................................................................................63 3.4. Conclusions ........................................................................................................................66 3.5. References ..........................................................................................................................77 CHAPTER 4. CHARACTERIZATION OF PQQ DERIVATIVES AND EVALUATION OF THEIR BIOLOGICAL ACTIVITIES........................................79 4.1. Introduction .......................................................................................................................79 4.2. Experimental......................................................................................................................81 4.3. Results and Discussion ......................................................................................................84 4.4. Conclusions ........................................................................................................................87 4.5. References ........................................................................................................................103 iv CHAPTER 5. PREPARATION AND CHARACTERIZATION OF HEMIN CONJUGATES FOR PROTEASE ASSAYS AND HOMOGENEOUS BINDING ASSAYS USING RECONSTITUTION OF APO-HORSERADISH PEROXIDASE AS A SIGNAL GENERATOR 5.1. Introduction .....................................................................................................................105 5.2. Experimental....................................................................................................................107 5.3. Results and Discussion ....................................................................................................112 5.4. Conclusions ......................................................................................................................116 5.5. References ........................................................................................................................131 CHAPTER 6. CONCLUSIONS AND FUTURE DIRECTIONS 6.1. Conclusions ......................................................................................................................133 6.2. Future Directions.............................................................................................................136 6.3. References ........................................................................................................................142 v LIST OF FIGURES FIGURE 1.1. Some biologically important redox potentials. 17 1.2. Structure of PQQ. 18 1.3. Crystal structure of the dimeric GDH with PQQ bound at the active center in the presence of calcium ions (black spheres) (The picture was generated using DeepView with the data of PDB ID: 1C9U). 19 1.4. Redox states of PQQ. 20 1.5. Proposed glucose oxidation mechanisms by sGDH: (A) hydride transfer (B) addition-elimination. 21 1.6. Structures of heme, hematin and hemin. 22 1.7. Crystal structure of HRP with heme bound at the active center (The picture was generated using DeepView with the data of PDB ID: 1W4W). 23 1.8. The working principle of homogeneous enzyme-linked immunoassay (EIA). 24 1.9. The working principle of ARIS. 25 1.10. Illustration of a “sandwich” type immunoassay system with a prosthetic group doped nanosphere as a label. 26 2.1. (a) GDH-PQQ reconstitution assay with DCPIP as an indicator (ox: oxidized form; red: reduced form). (b) Scheme of “sandwich” type immunoassay arrangement utilizing the new PQQ-doped PMMA nanoparticles as a tracer and biotin-avidin linkage to bind the nanoparticles to the reporter antibody of the assay. 45 2.2. (a) PQQ dose response in GDH-PQQ reconstitution assay in Tris-Sulfate buffer (insert: real time monitoring of DCPIP reduction at 590 nm). (b) PQQ dose response in GDH-PQQ reconstitution assay in Tris-Sulfate buffer containing 20% acetonitrile (insert: real time monitoring of DCPIP reduction at 590 nm). 46 vi 2.3. Digital picture of PQQ assay showing visual determination. 47 2.4. Preparation of PQQ-TDMA lipophilic salt. 48 2.5. Results of screening studies of different polymeric materials for encapsulating and releasing PQQ-TDMA salt into the polymer bulk phase. 49 2.6. SEM pictures of PMMA nanoparticles before and after PQQ loading. 50 2.7. Characterization of PMMA-PQQ particles by measuring released PQQ from an aliquot of 5 µL particles after 20 min incubation with Tris-Sulfate buffer and the