LIMITED PROTEOLYSIS OF STREPTOKINASE AND PROPERTIES OF SOME FRAGMENTS

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin w as performed under various incubation conditions and analysed by polya crylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th2 6, and Th16) were isolated and characterized further. The N-terminal s equences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr2 7 and Th26 were determined by partial sequencing. The evidence availab le allows the positioning of these fragments within the Sk sequence. F ragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is for med by a large polypeptide Ser60-Lys293 and non-covalently bonded smal ler polypeptides composed of amino acids from the N-terminal region Il e1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60- Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically- determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar compositio n were found in all the fragments studied using circular dichroism (c. d.) and infrared (i.r.) measurements. Differential scanning calorimetr ic (d.s.c.) measurements were performed in order to correlate the sequ ence regions of Sk to energetic folding units of the protein. Fragment s Sk1, Tr27, Th17, and Th16 show one melting peak in the temperature r ange from 42.8 to 46.1-degrees-C (thermal unfolding stage). For fragme nt Sk1, this melting peak can be separated by deconvolution into two t ransitions at T1 = 46.1-degrees-C and T2 = 47.3-degrees-C with DELTA-H -1 = 450 kJ/mol and DELTA-H-2 = 219 kJ/mol, respectively. Fragments Tr 17 and Th16 show one two-state transition at T = 42.8-degrees-C with D ELTA-H = 326 kJ/mol.