Activation of a tyrosine kinase receptor Met by hepatocyte growth factor (HGF) requires binding of proteolytically activated, two-chain (tc) HGF, but the biochemical detail of this ligand-receptor interaction specificity remains elusive because biologically inactive single chain (sc) HGF can also bind to Met with high affinity. We found that this proteolysis-independent Met binding can be eliminated by mutagenesis introduced in the kringle domain without losing the ability to bind and activate cellular Met receptor after proteolytic activation, arguing against this site's involvement in the physiological signalling. This non-signal producing Met-HGF interaction can also be eliminated by addition of a heparin mimetic sucrose octasulphate (SOS). By including SOS in the running buffer, we succeeded in detecting cleavage-dependent tcHGF-Met complex formation by size exclusion chromatography.