Chelation affects the conformation, lability and aggregation of channel catfish (Ictalurus punctatus) phosphorylcholine-reactive protein (PRP).
1. Phosphorylcholine-reactive protein (PRP) affinity-purified from channel catfish (Ictalurus punctatus) serum on phosphorylcholine-Sepharose, eluted from Bio-Gel A-5M as a 94.6 +/- 2.4 kDa protein when the gel filtration column buffer (Tris-saline) contained 25mM ethylenediaminetetraacetic acid (EDTA). 2. PRP chelated with EDTA immediately after affinity purification and gel-filtered in Tris-saline-EDTA, eluted as a 75.5 +/- 2.67 kDa protein referred to as fast-PRP (F-PRP). 3. PRP and F-PRP were identical on SDS-PAGE. Both resolved as a broad band of protein (ca 86-100 kDa) on non-reducing gels or as a ca 100 kDa protein after reduction with 2-mercaptoethanol (2-ME). 4. After gel-filtration in Tris-saline-EDTA, nearly complete reduction of 100 kDa PRP was achieved on SDS-PAGE. However, the protein regained its resistance to reduction upon storage at -60 degrees C. 5. SDS-PAGE and native PAGE also revealed that during storage, PRP and F-PRP combined to form 3 different aggregates referred to as aggregated-PRP (aggPRP). These aggregates are readily dissociated in the presence of 2-ME, suggesting a covalent interaction between adjacent pentamers comprising decameric aggPRPs. 6. PRP, F-PRP, and aggPRP have similar amino acid compositions.
Szalai
A J
AJ
Department of Microbiology, University of Mississippi Medical Center, Jackson 39216-4505.
Bly
J E
JE
Clem
L W
LW
D000818:Animals; D002097:C-Reactive Protein / Q000737:chemistry / Q000302:isolation & purification*; D002614:Chelating Agents; D002846:Chromatography, Affinity; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D007059:Ictaluridae / Q000097:blood*; D008970:Molecular Weight; D011487:Protein Conformation
D002614:Chelating Agents; D002097:C-Reactive Protein; D004492:Edetic Acid