Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations

S. Pal, Z. Chen, X. Xu, M. Mikhailova, B. Steffensen

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Background 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.

Original languageEnglish (US)
Pages (from-to)292-295
Number of pages4
JournalJournal of Periodontal Research
Volume45
Issue number2
DOIs
StatePublished - Apr 2010

Keywords

  • Cell attachment
  • Cell migration
  • Dimethyl sulfoxide
  • Fibronectin purification
  • Gelatinase
  • Human gingival fibroblast

ASJC Scopus subject areas

  • Periodontics

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