Limited proteolysis of streptokinase and properties of some fragments

• Published in: International journal of biological macromolecules Vol. 14; no. 2; pp. 107 - 116
• Main Authors: Misselwitz, R., Kraft, R., Kostka, S., Fabian, H., Welfle, K., Pfeil, W., Welfle, H., Gerlach, D.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.1992
• Subjects: Amino Acid Sequence; c.d.- and i.r.-spectroscopy; calorimetry; Chromatography; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; limited proteolysis; Molecular Sequence Data; Peptide Fragments - metabolism; Spectrometry, Fluorescence; Streptokinase; Streptokinase - chemistry; Streptokinase - metabolism; streptokinase fragments; Thermodynamics; Thermolysin; Trypsin; calorimetry; Streptokinase; c.d.- and i.r.-spectroscopy; limited proteolysis; streptokinase fragments
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/0141-8130(92)90007-U

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser 60-Lys 293 and non-covalent bonded smaller polypeptides composed of amino acids from the N-terminal region Ile 1-Lys 59 of Sk. Fragment Tr27 consists of the large polypeptide Ser 60-Lys 293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminalpolypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe 63-His 291. The N-termini of fragments Tr17 and Th16 start with Glu 148 and Ile 151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys 293 and His 291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1°C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transition at T 1 = 46.1° C and T 2 = 47.3° C with ΔH 1 = 450 kJ/ mol and ΔH 2 = 219 kJ/ mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8° C with ΔH = 326 kJ/ mol.