HPLC in Enzymatic Analysis
Second Edition
by Edward F. Rossomando

The role of enzymes in biological research has expanded, the application of HPLC and enzymes has extended to more disciplines, advances in separation techniques and instrumentation have increased the capability of HPLC, and the discovery of new enzymes has spawned new methods of analysis since the first edition was published.

High Performance Liquid Chromatography in Enzymatic Analysis, Second Edition addresses these developments in its coverage of the refinements of HPLC methods and their use in a wide range of laboratory applications. This book offers the same practical approach found in the first edition, incorporates a wealth of new information into existing chapters, and adds new chapters to deal with new applications, including capillary electrophoresis, forensic chemistry, microdialysis, and the polymerase chain reaction.

Topics

• Application of HPLC to the Assay of Enzymatic Activities
  
• Concepts and Principles of HPLC, Including the Latest Technological Advances
  
• Concepts and Principles of Capillary Electrophoresis (CE)
  
• Strategy for Design of an HPLC/CE System For Asssay of Enzyme Activity
  
• Preparation of Enzymatic Activities from Tissues and Single Cells
  
• Analysis of Enzymatic Activites in Body Fluids, Including Chromatobiosis
  
• HPLC for the Identification of New Enzymatic Activities
  
• Fundamentals of the Polymerase Chain Reaction
  
• HPLC in Forensics
  
• Survey of Enzymatic Activities Assayed by the HPLC Method, Including Many New Categories
  
• Multienzyme Systems, Including Many New Examples
  
• HPLC in the Analysis of Contaminated Food

Contents

1. Application of HPLC to the Assay of Enzymatic Activities
   
   • Anatomy of an Enzyme Assay
     
   • Classification of Enzymatic Assay Methods
     
   • Criteria for the Selection of an Assay Method
   
   
2. Concepts and Principles of High Performance Liquid Chromatography
   
   • Introduction of HPLC
     
   • Basic Components and Operation
     
   • Coupling the Components: On the Perils of Ferrules
     
   • Chromatogram
     
   • Interpretation of the Chromatogram
     
   • Selection of the Stationary Phase: Some Help From and Understanding of the Process of Separation
     
   • Composition and Preparation of the Mobile Phase
     
   • Column Maintenance
     
   • Monitoring Column Performance
   
   
3. Concepts and Principles of High Performance Capillary Electrophoresis
   
   • HPCE: Definition, History,and Literature
     
   • Basic Components and Operations
     
   • Process of Electrophoretic Separation
     
   • Instrumentation in Detail
     
   • Separation Efficiency and Resolution
     
   • Methods
   
   
4. Strategy for Design of an HPLC System for Assay of Enzyme Activity
   
   • Setting Up the Assay
     
   • Use of HPLC to Establish Optimal Conditions for the Enzymatic Reaction
   
   
5. Strategy for the Preparation of Enzymatic Activities from Tissues, Body Fluids, and Single Cells
   
   • Preparation and Assay of Enzymatic Activities in Samples of Tissues, Organs, and Bioligical Fluids
     
   • Preparation and Assay of Activities in Intact Cells
     
   • Preparation and Assay of Activities in Subcellular Samples
     
   • Initial Purification and Assay Of Activities in Cell-Free Lysates
     
   • HPLC for Purification of Enzymes: A Brief Background
     
   • Strategy for Use of HPLC in the Purification of Activities
     
   • Problems Related to the Assay of Activities Following Their Purification by HPLC
   
   
6. Microdialysis: An In Vivo Method for the Analysis of Body Fluids
   
   • Technical Aspects of Microdialysis
     
   • Applications of Microdialysis/HPLC in Enzymatic Analysis
     
   • Typical Analytes: Small Molecules
     
   • Estimating Enzymatic Activities
   
   
7. Fundamentals of the Polymerase Chain Reaction and Separation of the Reaction Products
   
   • Polymerase Chain Reaction
     
   • Principles of Nucleic Acid Separation
     
   • CE Methods: Principles and Stratagies for Nucleic Acids
     
   • Applications
   
   
8. Applications of HPLC/HPCE in Forensics
   
   • Forensic Toxicology
     
   • HPCE Analysis of Illicit Drug Substances
     
   • Analysis of Gunshot Residues and Constituents of Explosives
     
   • Analysis of Pen Inks
     
   • Analysis of Proteins Forensics
     
   • Analysis of Nonenzymatically Modified Proteins
     
   • Enzymatic Activity Assay by Capillary Electrophoresis
     
   • Protein-drug Binding Assays
     
   • Nucleic Acids and Their Constituents
   
   
9. Survey of Enzymatic Activities Assayed by the HPLC Method
   
   • Catecholamine Metabolism
     
   • Proteinase
     
   • Amino Acid and Peptide Metabolism
     
   • Polymine Metabolism
     
   • Heme Metabolism
     
   • Carbohydrate Metabolism
     
   • Steroid Metabolism
     
   • Purine Metabolism
     
   • Oxygenations
     
   • Pterin Metabolism
     
   • Lipid Metabolism
     
   • Modification of Proteins and Peptides
     
   • Vitamin Metabolism
     
   • Xenobiotic Metabolism
     
   • Pyrimidine Metabolism
     
   • Metabolism of Complex Saccharides and Glycoproteins
     
   • Miscellaneous
     
   • Nucleic Acid Modification and Expression
   
   
10. Multienzyme Systems
    
    • Assay of Two Activities Forming Different Products from the Same Substrate
      
    • Assay Of Two Activities Forming the Same Product for the Same Substrate
      
    • Formation of Two Separate Products from Two Separate Substrates by the Same Activity
      
    • Assay of Multienzyme Complex by the Reconstitution Method
      
    • Assay of a Multienzyme Complex Using the HPLC Method

Appendices
Index

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