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

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.

journal_name

J Periodontal Res

journal_title

Journal of periodontal research

authors

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

doi

10.1111/j.1600-0765.2009.01241.x

subject

Has Abstract

pub_date

2010-04-01 00:00:00

pages

292-5

issue

eissn

0022-3484

issn

1600-0765

pii

JRE1241

journal_volume

pub_type

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