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Pal S, Chen Z, Xu X, Mikhailova M, Steffensen B: Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations. J Periodontal Res. 2009 Nov 9.
Pal S, Chen Z, Xu X, Mikhailova M, Steffensen B. Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01241.x. (c) 2009 The Authors. Journal compilation (c) 2009 Blackwell MunksgaardBackground and Objective: Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN. Material and Methods: Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration. Results: Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly. Conclusion: The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN. |
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