Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen

Abstract

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 .mu.M, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 .mu.M and Kd = 220 .mu.M). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoic acid, pentylamine and arginine (Kd values are 3.2 .mu.M, 4.8 .mu.M and 4.3 .mu.M, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle.