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


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.


CONCLUSIONS
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.
Pal
S
S
Department of Periodontics and Biochemistry, University of Texas Health Science Center at San Antonio, TX 78229-3900, USA.
Chen
Z
Z
Xu
X
X
Mikhailova
M
M
Steffensen
B
B
D001669:Biochemical Phenomena; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D003362:Cost-Benefit Analysis; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004340:Drug Contamination / Q000517:prevention & control; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000187:drug effects; D005353:Fibronectins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D018093:Gelatinases / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D005881:Gingiva / Q000166:cytology / Q000187:drug effects; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry; D013997:Time Factors; D014508:Urea / Q000737:chemistry
D005353:Fibronectins; D014508:Urea; D018093:Gelatinases; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D004121:Dimethyl Sulfoxide