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 pM, contains two additional lysinebinding sites which differ in binding strength (Kd = 28 pM and Kd = 220 pM). 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 pM, 4.8 pM and 4.3 pM, 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.The nonprotease part of the plasminogen molecule contains five very similar modules of about 10 kDa each, known as kringles [l]. Kringles consist of approximately 80 amino acid residues which form structural domains that are stabilized by there disulphide bonds [l, 21. Kringle structures play a key role in protein-protein interaction of plasminogen and plasmin with components of the fibrinolytic system. These interactions are realized by means of the so-called lysinebinding sites localized in the kringles [3]. Lysine-binding sites are responsible for binding of plasminogen and its kringlecontaining fragments to lysine, 6-aminohexanoic acid and other o-aminocarboxylic acids [4].Recent studies by Markus and co-workers [5, 61 have shown that plasminogen has one strong and approximately four considerably weaker binding sites for 6-aminohexanoic acid and the antifibrinolytic agent tranexamic acid. It was established that lysine-binding sites are localized in kringles 1 and 4 [I, 7, 81. The high-affinity lysine-binding site is assumed to reside in kringle 1 [7, 81. At the same time it was considercd that kringle 5 does not contain such a site since miniplasminogen containing kringle 5 and isolated kringle 5 do not adsorb to lysine -Sepharose [I, 21. Nevertheless, we have shown that kringle 5 binds 6-aminohexanoic acid with a K,, of' 64 pM in solution [9]. This fact points out directly that kringle 5 also has a lysine-binding site. In this connection the natural necessity arises of more detailed investigation of kringle 1 + 2 + 3 with the aim of determining the number of binding sites localized in the first three kringles. One of the possible approaches for this is fluorescence spectroscopy. The application of this method is due to the fact that tryptophan residues at position 25,61 and 71 in kringle 4 and tryptophan residues at position 25 and 61 in kringle 1 are involved in lysine-binding site forma...
Affinity chromatography of plasminogen and its proteolytic fragments on immobilized fibrinogen TSD fragment has shown that the latter contains a plasminogen‐binding site which is complementary to the lysine‐binding site(s) of plasminogen molecule 1–3 kringle structures.
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