Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many 
substrates and is produced by most cell types as a zymogen, proMMP-9, in complex 
with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 
occurs as monomers, homomultimers and heterocomplexes, but our knowledge about 
the overall structure of proMMP-9 monomers and multimers is limited. We 
investigated biochemical, biophysical and functional characteristics of zymogen 
and activated forms of MMP-9 monomers and multimers. In contrast with a 
conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate 
that these are reduction-sensitive trimers. Based on the information from 
electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 
trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for 
TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent 
of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. 
Our results show that proMMP-9 trimers constitute a novel structural and 
functional entity that is differentially regulated by TIMP-1.