Fibrolase. A fibrinolytic protein from snake venom.
Fibrolase is an active fibrinolytic agent and possesses potential for use in thrombolytic therapy. Its mode of action had been characterized, both in vitro and in vivo. Possessing three disulfide bonds, native fibrolase is nonglycosylated and binds an intrinsic zinc atom. The zinc is essential for retention of activity and structural integrity. In solution, fibrolase is sensitive to changes in pH and temperature (Pretzer et al., 1991). At neutral to basic pH (pH 5-9), the solubility and stability of fibrolase is nearly constant. Little structural variation can be detected by CD spectroscopy. However, decrease in pH below 5 leads to a pronounced reduction in both the solubility and activity of fibrolase. At pH 3 and below, the solubility of fibrolase returns but the activity does not. This solubility profile is unusual in that the minimal solubility is well removed from the pI (which is 6.7). It is proposed that the behavior of fibrolase with variation in pH can be understood in terms of capacity to bind zinc. At pH 5 to 9, the protein binds zinc and the structure and activity are preserved. Near pH 5, the histidine residues which serve as ligands for the zinc become protonated and zinc binding is lost. Loss of zinc leads to local unfolding of a helical segment of fibrolase, exposing hydrophobic groups which allow the protein to rapidly aggregate. At lower pH values (1-3), the protein again adopts a more globular structure, similar to molten globule states, and the solubility increases. However, without the zinc, fibrolase remains inactive. Changes in pH also affect thermal stability. The Tm for fibrolase moves from 50 degrees C at pH 8 to 43 degrees C at pH 5. Increases in temperature also lead to removal of the zinc ion, again producing a partially denatured protein with a marked tendency to aggregate. In both cases (decrease in pH and increase in temperature), analysis of the CD spectra indicates that the protein has primarily lost alpha-helical secondary structure. A major change in structure can also be observed using NMR spectroscopy. At temperatures below 35 degrees C, the globular structure of fibrolase remains intact, although some increase in chain mobility can be noted with increased temperature. Upon melting, numerous signals collapse as the protein unfolds. Transition temperatures (Tm) as measured by CD and NMR are in good agreement. Similar structural changes can be induced by adding zinc chelators such as EDTA and DTT. This leads to complete loss of activity at EDTA concentrations above 1.0 mM.(ABSTRACT TRUNCATED AT 400 WORDS)
Merck Research Laboratories, West Point, Pennsylvania 19486.
D
D
Pretzer
B S
BS
Schulteis
C D
CD
Smith
D G
DG
Vander Velde
J W
JW
Mitchell
M C
MC
Manning
D000595:Amino Acid Sequence; D000818:Animals; D004355:Drug Stability; D004795:Enzyme Stability; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D012910:Snake Venoms / Q000201:enzymology*
D012910:Snake Venoms; D008666:Metalloendopeptidases; C058789:fibrolase