FUNCTIONAL INDEPENDENCE OF THE KRINGLE-4 AND KRINGLE-5 REGIONS OF HUMAN PLASMINOGEN

As part of continuing studies to evaluate whether the kringle domain regions of human plasminogen (HPg) exhibit independent conformational properties, simple model systems are required. Toward this end, we have constructed cDNA regions of HPg encoding its kringle 4 ([K4HPg]) and kringle 4-5 ([K4HPgK5HPg]) regions, expressed these gene fragments in bacterial cells, and purified the recombinant (r) products. The resulting r-[K4HPgK5HPg] was also employed to obtain the r- [K5HPg] domain of HPg by limited elastolytic digestion of this double-kringle polypeptide. The omega-amino acid ligand binding properties and thermal denaturation characteristics of r-[K4HPg], r-[K5HPg], and r-[K4HPgK5HPg] were determined, along with those for the [K5HPg] domain linked to the protease (P) region of HPg ([K5HPg]P). This allowed us to evaluate whether the conformational properties of the [K5HPg] module were influenced by the presence of its neighboring domains in HPg. The temperature midpoint of maximum heat capacity, T(m) (and calorimetric enthalpy, DELTAH), for thermal denaturation of r-[K4HPg] was 57.8-degrees-C (79.8 kcal/mol) in the absence of epsilon-aminocaproic acid (EACA) and 70.8-degrees-C (93.7 kcal/mol) in the presence of that ligand. The corresponding values for isolated r-[K5HPg] were 50.4-degrees-C (78.4 kcal/mol) and 61.0-degrees-C (89.8 kcal/mol), respectively. These parameters for the isolated kringles were essentially unchanged when these same kringle domains were present in the r-[K4HPgK5HPg] and [K5HPg] P covalently linked pairs. Similarly, the thermodynamic characteristics (DELTAG, DELTAH, and DELTAS) that describe the binding energy of EACA to r-[K4HPg] at 25-degrees-C were -6.3 kcal/mol, -4.5 kcal/mol, and 6.0 eu, respectively. Those for similar binding to r-[K5HPg] were -5.3 kcal/mol, -5.8 kcal/mol, and -1.7 eu. Again, no major differences were found in these binding properties for the domains when present in the paired domains, r-[K4HPgK5HPg] and [K5HPg]-P. These results support the conclusion that these two kringle regions of HPg likely exist as independently folded modules. Thus, meaningful investigations can take place on the structure-function relationships of these kringle domains when isolated from the intact protein. This situation eases the interpretation of results of investigations using intact HPg and allows the assignment of protein properties to discrete regions of the molecule.