Heat-induced fragmentation of human plasma fibronectin

Human plasma fibronectin was found to undergo fragmentation during heat-denaturation, leading to artifacts in SDS-polyacrylamide gel electrophoretic analyses. Electrophoretic patterns of heated samples showed a progressive decrease in intact fibronectin chains (225 kDa) which coincided with the appearance of increasing amounts of numerous smaller components having molecular weights ranging from 10 000 to 200 000. The fragmentation was temperature-dependent, being undetectable after 2 h at 60 degrees C, but detectable after 30 min at 70 degrees C or as little as 2 min at 100 degrees C. After 2 h at 100 degrees C, the intact monomer was no longer visible. Neither mercaptoethanol nor SDS was required for fragmentation. Sterile filtration or pretreatment with inhibitors of proteolytic enzymes had no effect. Treatment with amines did not diminish the degradation, indicating that the process differs from heat-fragmentation of alpha 2-macroglobulin and complement proteins, which occurs at a reactive internal thiolester bond. Fibronectin fragmentation was highly pH-dependent, being markedly accelerated under acidic conditions, suggesting that autolytic cleavage of the peptide chain at acid-labile aspartyl bonds was responsible for this phenomenon.