UC San Diego UC San Diego Electronic Theses and Dissertations Title A whole blood protease assay Permalink https://escholarship.org/uc/item/1r6885gb Author Lefkowitz, Roy Brian Publication Date 2010 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO A Whole Blood Protease Assay A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Roy Brian Lefkowitz Committee in charge: Professor Michael J. Heller, Chair Professor Sadik C. Esener Professor David Gough Professor Geert W. Schmid-Schönbein Professor Mark A. Talamini 2010 Copyright Roy Brian Lefkowitz, 2010 All rights reserved 2 The Dissertation of Roy Brian Lefkowitz is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2010 iii DEDICATION To my mother, my late father, my brother, and my wife, Thank you very much for all of your love and support. I owe my success to all of you. iv EPIGRAPH “I've failed over and over and over again in my life and that is why I succeed.” Michael Jordan v TABLE OF CONTENTS Signature Page ................................................................................................................... iii Dedication.......................................................................................................................... iv Epigraph...............................................................................................................................v Table of Contents...............................................................................................................vi List of Figures......................................................................................................................x List of Tables .................................................................................................................... xii List of Abbreviations ....................................................................................................... xiii Acknowledgements............................................................................................................xv Vita ....................................................................................................................................xx Abstract of the Dissertation ........................................................................................... xxiii Chapter 1: Introduction to Protease Detection.....................................................................1 1.1 Motivation for Protease Detection.............................................................................1 1.2 Proteases in Physiological Shock ..............................................................................4 1.3 Proteases in Other Diseases.......................................................................................6 1.4 Previous Detection and Concentration Strategies ...................................................10 1.4.1 Detection of Activity ........................................................................................10 1.4.2 Detection of Concentration Levels ...................................................................14 1.4.3 Sample Concentration Strategies......................................................................17 1.5 The Issue with Sample Preparation .........................................................................21 1.6 A Whole Blood Protease Assay...............................................................................23 Chapter 2: Charge-Changing Substrates for Whole Blood Protease Detection ................31 2.1 Introduction .............................................................................................................31 2.2 Materials and Methods ............................................................................................32 2.2.1 Materials ...........................................................................................................32 2.2.2 Detection Limit in 1X PBS and Plasma............................................................33 vi 2.2.3 Detection Limit in Whole Blood ......................................................................34 2.2.4 Specificity .........................................................................................................35 2.2.5 Kinetics .............................................................................................................36 2.2.6 Trypsin Detection in Multiple Gel Formats......................................................36 2.3 Results and Discussion ............................................................................................37 2.3.1 Detection Limit in 1X PBS...............................................................................38 2.3.2 Detection Limit in Human Plasma....................................................................39 2.3.3 Detection Limit in Whole Blood ......................................................................40 2.3.4 Substrate Specificity and Kinetics ....................................................................41 2.3.5 Trypsin Detection in Multiple Gel Formats......................................................42 2.4 Concluding Remarks ...............................................................................................44 2.5 Acknowledgements .................................................................................................44 Chapter 3: Polyanionic Focusing Gel Electrophoresis ......................................................53 3.1 Introduction .............................................................................................................53 3.2 Materials and Methods ............................................................................................55 3.2.1 Materials ...........................................................................................................55 3.2.2 Detection in 1X PBS using Various Classical Gel Formats .............................56 3.2.3 Detection in 1X PBS using Focusing Gels .......................................................57 3.2.4 Time Course of Signal Enhancement in Focusing Gel.....................................58 3.2.5 Detection in Whole Blood and Plasma with Focusing Gels.............................59 3.2.6 Detection of Spiked Trypsin in Whole Blood ..................................................60 3.3 Results and Discussion ............................................................................................61 3.3.1 Detection in 1X PBS using Various Classical Gel Formats .............................62 3.3.2 Detection in 1X PBS using Focusing Gels .......................................................64 3.3.3 Time Course of Signal Enhancement in a Focusing Gel..................................66 3.3.4 Detection in Whole Blood and Plasma with Focusing Gels.............................67 vii 3.3.5 Detection of Spiked Trypsin in Whole Blood ..................................................69 3.4 Concluding Remarks ...............................................................................................71 3.5 Acknowledgements .................................................................................................72 Chapter 4: Substrates for Elastase, Chymotrypsin, MMP-2, and MMP-9 ........................84 4.1 Introduction .............................................................................................................84 4.2 Materials and Methods ............................................................................................86 4.2.1 Materials ...........................................................................................................86 4.2.2 Substrate Specificity .........................................................................................87 4.2.3 Detection in 1X PBS using 25% Polyacrylamide Gels ....................................88 4.2.4 Detection in 1X PBS using Focusing Gels .......................................................89 4.2.5 Detection of Spiked Protease in Whole Blood .................................................90 4.3 Results and Discussion ............................................................................................91 4.3.1 Substrate Specificity .........................................................................................92 4.3.2 Detection in 1X PBS using 25% Polyacrylamide Gels ....................................96 4.3.3 Detection in 1X PBS using Focusing Gels .......................................................97 4.3.4 Detection of Spiked Protease in Whole Blood ...............................................100 4.4 Concluding Remarks .............................................................................................103 4.5 Acknowledgements ...............................................................................................104 Chapter 5: Measurement of Protease Activity in Disease ...............................................113 5.1 Introduction ...........................................................................................................113 5.2 Materials and Methods ..........................................................................................118 5.2.1 Materials .........................................................................................................118