Denaturation-renaturation of the fibrin-stabilizing factor XIII a-chain isolated from human placenta. Properties of the native and reconstituted protein.
The denaturation-renaturation transition between the native and unfolded states of the dimeric blood coagulation factor XIIIa has been examined by far-UV circular dichroism, fluorescence spectroscopy, activity measurements, sedimentation equilibrium analysis, and size exclusion high performance liquid chromatography. Guanidine hydrochloride and urea-dependent denaturation in the absence and in the presence of 5mM dithioerythritol or glutathione (5mM GSH) exhibit biphasic transitions. The first stage represents a sharp transition characterized by a change in secondary structure without subunit dissociation. This step is accompanied by the irreversible loss of biological activity. The second transition reflects the dissociation and complete unfolding of the protein to a random coil. After loss of biological activity no reactivation can be accomplished under any of the following conditions: (i) denaturation and renaturation under reducing or non-reducing conditions, (ii) variation of the protein concentration and temperature, (iii) addition of specific ligands (Ca2+, substrate), (iv) presence of stabilizing and/or destabilizing agents. Attempts to renature the protein under standard conditions (0.1 M Tris/HCl pH 7.5-9.0, 5mM DTE, 5mM EDTA) lead to refolding intermediates which exhibit a strong tendency to aggregate. A soluble product of reconstitution can be obtained by refolding at low protein concentration, low temperature, and in the presence of small amounts of destabilizing agents such as arginine or urea in the renaturation buffer at pH 7.5 to 9. The spectroscopic and hydrodynamic characterization of the partially reconstituted (non-native inactive) protein shows that partially reconstituted factor XIIIa exhibits the fluorescence properties and the dimeric structure of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Rinas
U
U
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg.
Risse
B
B
Jaenicke
R
R
Abel
K J
KJ
Zettlmeissl
G
G
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004793:Enzyme Reactivators; D005260:Female; D006801:Humans; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D011487:Protein Conformation; D011489:Protein Denaturation; D011503:Transglutaminases / Q000032:analysis / Q000378:metabolism*
D004793:Enzyme Reactivators; D011503:Transglutaminases