Kc. Ingham et al., BINDING OF HEPARIN BY TYPE-III DOMAINS AND PEPTIDES FROM THE CARBOXY-TERMINAL HEP-2 REGION OF FIBRONECTIN, Biochemistry, 32(46), 1993, pp. 12548-12553
The major sites of heparin binding by fibronectin are located in fragm
ents of 30 or 40 kDa that contain type III modules 12 through 14 or 15
. Various proteolytic or recombinant subfragments and several syntheti
c peptides derived from this region have been compared with respect to
their binding to fluorescein-labeled heparin in solution. Binding was
monitored by the change in fluorescence anisotropy at 25-degrees-C an
d pH 7.4 in 0.02 M Tris buffer, alone (TB) or with 0.15 M NaCl (TBS).
A 23-kDa fragment containing III13 and III14 but lacking III12 had K(d
) values of 0.3 and 1.8 muM in TB and TBS, respectively, indistinguish
able from the 30-kDa parent. Fragments containing only module III13 bo
und 2-3-fold weaker than the parent while those containing only III14
bound 6-50-fold weaker depending on the ionic strength. Fragments cont
aining only III12 or III15 failed to bind at all in TBS. A cationic pe
ptide derived from the amino terminus of III13 and containing the Arg-
Arg-Ala-Arg consensus sequence, whose integrity was shown by Barkalow
and Schwarzbauer [Barkalow, F. J., & Schwarzbauer, J. E. (1991) J. Bio
l. Chem. 266, 7812-7818] to be critical, failed to bind in TBS but bou
nd weakly in TB. Two additional cationic peptides derived from the mid
dle and C-terminal regions of III14 showed similar behavior. Thus whil
e the major determinant(s) of heparin binding are located in III13, th
ose determinants are only active when part of a properly folded struct
ure. Furthermore,module III13 when isolated had a slightly lower affin
ity than fragments containing both III13 and III14. It is concluded th
at interactions between these two modules may be important to arrange
positively charged residues from both modules for optimal recognition
by heparin.