Author/Editor		Trampuš-Bakija, Alenka; Stegnar, Mojca; Stojan, Jure
Title		Amidolitična metoda za merjenje aktivnosti trombina: primerjava klasične encimske analize in analize progresivnih krivulj
Translated title		Amidolytic assay for thrombin activity determination: comparison of classical enzyme analysis and progress curves analysis
Type		članek
Source		Med Razgl
Vol. and No.		Letnik 39, št. Suppl 5
Publication year		2000
Volume		str. 63-8
Language		slo
Abstract		Thrombin plays a central role in the coagulation process. Development and synthesis of new antithrombotic agents and synthetic thrombin inhibitors is an area of intense research. Thrombin activity and/or inhibitory potency of reversible competitive thrombin antagonists can be determined using the amidolytic enzyme assay. In our experiments we have chosen concentrations of the enzyme and the substrate suitable for the determination of thrombin inhibitors with inhibition constants between 0.1 and 10 micro M. In order to follow the colored product formation in the reaction between thrombin and chromogenic substrate 5-2238, we used classical spectrophotometer and stopped-flow apparatus. Classical kinetic analysis of initial reaction rates, obtained at various substrate concentrations, resulted in a Michaelis constant Km of 16 micro M. Using stopped flow technique, we measured progress curves showing complete hydrolysis of the substrate into the products. The analysis was performed by fitting the system of differential equations for several possible kinetic mechanisms to the complete course of all experimental progress curves simultaneously. Among the possible models the most suitable one was the model allowing for product inhibition. Since the structure of colorless tripeptide product resembles the potent competitive thrombin inhibitors such a result is obvious and expected. The Km determined in this way, however, was one grade of magnitude lower, being 2.8 micro M. It seems that the two results differ because of unreliable data provided by the classical method at low substrate concentrations. These data are essential for the determination of Km. On the other hand, the progress curves obtained by the stopped-flow technique are reliable, repeatable and significantly more informative. Therefore, much fewer measurements are necessary to obtain more reliable results.
Summary		Trombin ima osrednjo vlogo pri procesu strjevanja krvi. Načrtovanje in sinteza novih sintetskih trombinskih zaviralcev, ki bi jih uporabljali za zdravljenje in preprečevanje tromboze, je obetavno področje. Za ugotavljanje aktivnosti trombina in v prihodnosti, učinkovitosti reverzibilnih kompetitivnih inhibitorjev trombina, smo izbrali amidolitično encimsko metodo. Koncentracije encima in substrata smo izbrali tako, da bomo lahko zasledovali inhibitorje s konstanto inhibicije med 0,11 micro M in 10 micro M. Časovni potek nastajanja obarvanega produkta v reakciji med trombinom in kromogenim substratom S-2238 smo spremljali fotometrično s klasičnim spektrofotometrom in z aparatom stopped-flow. S klasično kinetično metodo smo izmerili začetne hitrosti reakcij pri različnih koncentracijah substrata in grafično določili Michaelisovo konstanto Km (16 pM). S stopped-flow tehniko pa smo izmerili progresivne krivulje, ki kažejo popolno hidrolizo substrata v produkt. Analizo smo izvedli tako, da smo sistem diferencialnih enačb za več možnih kinetičnih mehanizmov prilagajali sočasno na celoten potek vseh izmerjenih progresivnih krivulj. Izmed možnih modelov je bil najustreznejši tisti, ki je upošteval produktno inhibicijo. Ker struktura tripeptidnega dela substrata spominja na inhibitorje trombina, je tak rezultat razumljiv in pričakovan. Na ta način določena Km pa je za cel velikostni razred nižja (2,81micro M). Zdi se, da se rezultata razlikujeta zaradi nezanesljivosti klasične metode, predvsem pri določanju začetnih hitrosti v prisotnosti nizkih koncentracij substrata. Podatki v tem območju namreč najbolj vplivajo na določitev Km. Po drugi strani pa so progresivne krivulje, izmerjene s stopped-flow tehniko, zelo zanesljive, ponovljive in dajo bistveno več informacij. Število meritev, potrebnih za določitev kinetičnih parametrov encimske reakcije, se zato pri analizi progresivnih krivulj v primerjavi s klasično analizo bistveno zmanjša.
Descriptors		THROMBIN ANTITHROMBINS KINETICS SPECTROPHOTOMETRY