Separation by heparin-affinity chromatography of catalytically active and inactive forms of trypsin which retain the (Na-K)ATPase stimulating property.

The possibility that different structural determinants on trypsin, other than catalytic sites, are involved in the cell membrane (Na-K)ATPase stimulating property was investigated by submitting bovine trypsin to two purification procedures: gel filtration on Sephadex G-50 and heparin-Sepharose chromatography. The latter procedure was also chosen in consideration of the known affinity for heparin displayed by serine proteinases. Trypsin peaks eluted from both columns were analysed by measuring esterolytic and proteolytic activities, the beef heart (Na-K)ATPase stimulating property and amino acid content. Fluorescence emission spectra and both non-denaturing and SDS-gel electrophoresis were also performed to test structural modifications on trypsin peaks. Four peaks eluted from Sephadex G-50 with variable estero-proteolytic and (Na-K)ATPase stimulating activities; the latter was also present in two peaks which displayed the lowest estero-proteolytic activities. All peaks proved to be trypsin in amino acid composition. Two peaks eluted from the heparin-Sepharose column with distinct biological activities: a first minor peak, eluted with the void volume, was catalytically inactive but it retained the (Na-K)ATPase stimulating activity. The second, major peak eluted mostly with 0.5 mol/l NaCl, displayed only esteroproteolytic activities, but no (Na-K)ATPase-stimulating activity. It overlapped control trypsin in both electrophoretic patterns, fluorescence emission spectrum and amino acid composition. The first peak showed differences with the parent compound, as revealed by the amino acid composition and tryptophan fluorescence emission spectrum. Marked differences were also observed in the electrophoretic pattern which only showed bands of low molecular mass mostly confined to the anode. NH2-terminus analysis confirmed that the first peak contained trypsin fragments originated from the parent compound after passage through the heparin column. It is hypothesized that trypsin binding to heparin causes structural alteration of the proteinase and primes the catalytic cleavage of fragments which lose heparin affinity and elute in the void volume. The results also confirm that the proteolytic mechanism is not involved in trypsin-mediated (Na-K)ATPase stimulation and indicate that heparin-Sepharose chromatography is a useful tool to separate catalytically active and inactive forms of trypsin.