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Untersuchungen Zur Proteolytischen Aktivität Pflanzlicher Milchsäfte Und Deren Einfluss Auf Die Hämostase Und Fibrinolyse

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Untersuchungen Zur Proteolytischen Aktivität Pflanzlicher Milchsäfte Und Deren Einfluss Auf Die Hämostase Und Fibrinolyse Untersuchungen zur proteolytischen Aktivität pflanzlicher Milchsäfte und deren Einfluss auf die Hämostase und Fibrinolyse Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Apotheker Martin Flemmig aus Mölln 2015 Die vorliegende Arbeit entstand in der Zeit von Oktober 2010 bis Mai 2015 unter der Leitung von Herrn Prof. Dr. Matthias F. Melzig am Institut für Pharmazie der Freien Universität Berlin. Erster Gutachter: Prof. Dr. Matthias F. Melzig Zweiter Gutachter: apl. Prof. Dr. Harshadrai M. Rawel Disputation am: 22.06.2015 Inhaltsverzeichnis Inhaltsverzeichnis Abkürzungsverzeichnis .......................................................................................................... IV 1. Einleitung ............................................................................................................................. 1 1.1 Milchsaft – Latex .......................................................................................................... 1 1.2 Euphorbiaceae JUSS. ...................................................................................................... 2 1.3 Proteasen ....................................................................................................................... 3 1.3.1 Einsatz von Proteasen als Therapeutika ............................................................. 3 1.3.2 Einsatz von Proteasen in anderen Bereichen ...................................................... 4 1.3.3 Funktion der im Milchsaft enthaltenen Proteasen .............................................. 5 1.3.4 Isolierte Proteasen aus Euphorbiaceae................................................................ 6 1.4 Hämostase und Fibrinolyse ........................................................................................... 7 1.4.1 Fibrin(ogen) ........................................................................................................ 8 1.4.2 Plasmin(ogen) ..................................................................................................... 9 1.4.3 Plasminogen-Aktivatoren ................................................................................. 10 1.4.4 Andere fibrinolytische Proteasen ...................................................................... 14 1.5 Zielsetzung .................................................................................................................. 16 2. Material .............................................................................................................................. 17 2.1 Geräte .......................................................................................................................... 17 2.1.1 Chromatographie .............................................................................................. 17 2.1.2 Elektrophorese .................................................................................................. 17 2.1.3 MALDI-TOF-MS ............................................................................................. 18 2.1.4 Sonstige Geräte ................................................................................................. 18 2.2 Chemikalien und Substanzen ...................................................................................... 19 2.2.1 Allgemein ......................................................................................................... 19 2.2.2 Kits .................................................................................................................... 20 2.2.3 Elektrophorese .................................................................................................. 20 2.2.4 Protease-Inhibitoren .......................................................................................... 22 2.2.5 Proteasen ........................................................................................................... 22 2.3 Verbrauchsmaterial ..................................................................................................... 22 2.3.1 Allgemeine Verbrauchsmaterialien .................................................................. 22 2.3.2 Elektrophorese .................................................................................................. 23 2.4 Biologisches Material ................................................................................................. 23 3. Methoden ............................................................................................................................ 25 3.1 Gewinnung von Latex ................................................................................................. 25 3.2 Gewinnung von Plasma .............................................................................................. 25 3.3 Protein – Gehaltsbestimmungen ................................................................................. 26 3.3.1 BCA – Assay .................................................................................................... 26 I Inhaltsverzeichnis 3.3.2 Bradford Assay ................................................................................................. 27 3.4 Protease – Aktivitätsbestimmungen ............................................................................ 27 3.4.1 Allgemeine proteolytische Aktivität ................................................................. 27 3.4.2 Aktivität gegenüber einem spezifischen Peptidsubstrat für Plasmin ................ 27 3.4.3 Plasminogen-Aktivator Aktivität ...................................................................... 28 3.5 Trübungsmessungen mit Fibrin(ogen) ........................................................................ 29 3.5.1 Gerinnung von Fibrinogen ................................................................................ 29 3.5.2 Gerinnungshemmung durch Fibrin(ogen)olyse ................................................ 29 3.6 Trübungsmessungen mit Plasma ................................................................................. 30 3.6.1 Calciumunabhängige Gerinnungsauslösung ..................................................... 30 3.6.2 Beeinflussung der Recalcifizierungszeit........................................................... 30 3.7 Fibrin(ogen)-Platten-Tests .......................................................................................... 31 3.7.1 Fibrinogen-Agarose-Platte ................................................................................ 31 3.7.2 Fibrin-Agarose-Platte ....................................................................................... 31 3.8 Auflösung von Plasma-Gerinnseln ............................................................................. 31 3.9 Elektrophorese ............................................................................................................ 32 3.9.1 Polyacrylamid-Gelelektrophorese .................................................................... 32 3.9.2 Bestimmung der Molekülmassen ..................................................................... 34 3.9.3 Isoelektrische Fokussierung.............................................................................. 34 3.9.4 Bestimmung des Fibrinogen-Abbaus ............................................................... 36 3.9.5 Zymographie ..................................................................................................... 36 3.9.6 Proteinfärbung .................................................................................................. 38 3.10 MALDI-TOF Massenspektrometrie ........................................................................... 39 3.10.1 Molekülmassenbestimmung und Reinheitsprüfung ......................................... 39 3.10.2 In-Gel Verdau ................................................................................................... 40 3.11 Isolation von Mauritanicain ........................................................................................ 43 3.11.1 Probenvorbereitung........................................................................................... 43 3.11.2 Ionenaustausch-Chromatographie .................................................................... 43 3.11.3 Ultrafiltration .................................................................................................... 44 3.11.4 Größenausschluss-Chromatographie ................................................................ 44 3.12 Bestimmung der Protease-Klasse ................................................................................ 45 3.13 Bestimmung der Temperaturabhängigkeit .................................................................. 45 3.14 Bestimmung der pH Abhängigkeit ............................................................................. 46 4. Ergebnisse und Diskussion ............................................................................................... 47 4.1 Screening ..................................................................................................................... 47 4.1.1 Allgemeine proteolytische Aktivität ................................................................. 47 4.1.2 Aktivität gegenüber einem spezifischen fluorogenen Substrat für Plasmin ..... 48 4.1.3 Vergleich der Zusammensetzung durch Elektrophorese .................................. 56 4.2 Isolation von Mauritanicain .......................................................................................
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