title: Stability of the thrombolytic protein fibrolase: effect of temperature and pH on activity and conformation.
abstract: The effect of temperature and pH on the activity and conformation of the thrombolytic protein fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (approximately 22 degrees C). At 37 degrees C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alpha-helical structure was lost in a cooperative transition (Tm of 50 degrees C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the Tm determined by NMR was 46 degrees C at pD 8. The effect of pH on the proteolytic activity and structure of fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of fibrolase decreased to 43 degrees C at pH 5. Protein concentrations determined over the pH range of 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.
authors.affiliation: Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: D
authors.initials: D
authors.lastname: Pretzer
authors.forename: B S
authors.initials: BS
authors.lastname: Schulteis
authors.forename: C D
authors.initials: CD
authors.lastname: Smith
authors.forename: D G
authors.initials: DG
authors.lastname: Vander Velde
authors.forename: J W
authors.initials: JW
authors.lastname: Mitchell
authors.forename: M C
authors.initials: MC
authors.lastname: Manning
mesh_terms: D002942:Circular Dichroism; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000494:pharmacology; D011487:Protein Conformation; D013696:Temperature
chemical_list: D008666:Metalloendopeptidases; C058789:fibrolase