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Inhibition of Zinc-Dependent Peptidases by Maillard Reaction Products

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Inhibition of Zinc-Dependent Peptidases by Maillard Reaction Products Inhibition of zinc-dependent peptidases by Maillard reaction products DISSERTATION zur Erlangung des akademischen Grades Doktor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Leticia Missagia de Marco geboren am 23. März 1978 in Belo Horizonte, Brasilien Eingereicht am: 09.10.2014 Verteidigt am: 09.03.2015 Die Dissertation wurde in der Zeit von April 2005 bis Dezember 2011 an der Professur für Lebensmittelchemie angefertigt. List of Contents List of Contents LIST OF CONTENTS .............................................................................................................. I LIST OF TABLES ................................................................................................................. IV LIST OF FIGURES ................................................................................................................. V LIST OF ABBREVIATIONS .............................................................................................. VII 1 INTRODUCTION ............................................................................................................ 1 2 BACKGROUND ............................................................................................................... 3 2.1 Maillard reaction in food ................................................................................................. 3 2.1.1 Melanoidins .................................................................................................................... 8 2.2 Coffee ............................................................................................................................... 11 2.2.1 General aspects ............................................................................................................ 11 2.2.1.1 Coffee production .................................................................................................... 12 2.2.1.2 General chemical composition ................................................................................ 14 2.2.1.3 Coffee and health .................................................................................................... 20 2.2.2 Coffee melanoidins ...................................................................................................... 24 2.2.2.1 Chemistry of coffee melanoidins ............................................................................ 24 2.2.2.2 Properties of coffee melanoidins ............................................................................. 29 2.3 Zinc metallopeptidases ................................................................................................... 32 2.3.1 Matrix metalloproteinases (MMPs) ............................................................................. 33 2.3.1.1 Functions of MMPs ................................................................................................. 35 2.3.1.2 Structure of MMPs .................................................................................................. 37 2.3.1.3 Inhibition of MMPs ................................................................................................. 39 2.3.2 Clostridium histolyticum collagenase (ChC) ............................................................... 43 2.3.2.1 Functions of ChC .................................................................................................... 43 2.3.2.2 Structure of ChC ..................................................................................................... 43 2.3.2.3 Inhibition of ChC .................................................................................................... 44 2.3.3 Agiotensin converting enzyme (ACE) ......................................................................... 45 2.3.3.1 Functions of ACE .................................................................................................... 45 2.3.3.2 Structure of ACE ..................................................................................................... 46 2.3.3.3 Inhibition of ACE .................................................................................................... 48 3 EXPERIMENTAL SECTION ....................................................................................... 50 3.1 Chemicals, materials and equipment ............................................................................ 50 3.1.1 Chemicals ..................................................................................................................... 50 3.1.2 Material ........................................................................................................................ 52 3.1.3 Equipment .................................................................................................................... 52 3.1.4 Solutions....................................................................................................................... 54 I List of Contents 3.2 Synthesis of Nα-carboxyalkylated peptides ................................................................... 55 3.2.1 Nα-carboxyalkylation of GP, LL, IA, GA, GL, AP, IP and IPP by reductive alkylation ............................................................................................................................................... 55 3.2.2 Nα-carboxyalkylation of IW using sodium cyanoborohydride .................................... 56 3.3 Purification ...................................................................................................................... 57 3.3.1 Ion Exchange Chromatographic purification ............................................................... 57 3.3.1.1 Spotting test ............................................................................................................. 58 3.3.2 HPLC purification of CM-IW ...................................................................................... 58 3.3.3 Overview of the synthesis and elution conditions ....................................................... 59 3.4 Characterization of carboxyalkylated peptides ........................................................... 61 3.4.1 Mass spectrometry ....................................................................................................... 61 3.4.2 Elemental Analysis ...................................................................................................... 61 3.4.3 Analytical characteristics of carboxyalkylated peptides .............................................. 62 3.5 Preparation of coffee fractions ...................................................................................... 65 3.5.1 Roasting conditions ...................................................................................................... 65 3.5.2 Fractionation of coffee samples: Isolation of coffee melanoidins ............................... 67 3.6 Structural studies ............................................................................................................ 69 3.6.1 Estimation of the molecular weight ............................................................................. 69 3.6.2 C/N ratio ....................................................................................................................... 70 3.6.3 Amino acid analysis ..................................................................................................... 70 3.6.3.1 Acid hydrolysis ....................................................................................................... 70 3.6.3.2 General amino acid analysis .................................................................................... 70 3.6.3.3 Lysinoalanine .......................................................................................................... 71 3.6.3.4 Pentosidine .............................................................................................................. 72 3.6.4 Total phenols ................................................................................................................ 74 3.6.5 Raman spectroscopy .................................................................................................... 74 3.7 Study on inhibition of zinc metalloproteases ................................................................ 75 3.7.1 Inhibition of ACE......................................................................................................... 75 3.7.1.1 General enzymatic assay ......................................................................................... 75 3.7.1.2 Quantification .......................................................................................................... 78 3.7.2 Inhibition of MMP-1, -2 and -9 ................................................................................... 79 3.7.2.1 General enzymatic assay ......................................................................................... 80 3.7.2.2 Effect of zinc addition on the inhibition of MMP-1 by melanoidins ...................... 81 3.7.3 Inhibition of ChC ......................................................................................................... 82 3.7.3.1 General enzymatic assay ......................................................................................... 82 3.7.3.2 Quantification .......................................................................................................... 84 3.7.4 Calculation of IC50 ......................................................................................................
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