Human matrix metalloproteinase 7 (MMP-7) is the smallest matrix metalloproteinase. It plays important roles in tumour invasion and metastasis. 8-Anilinonaphthalene 1-sulphonate (ANS) is a fluorescent probe widely used for the analysis of proteins. It emits large fluorescence energy when its anilinonaphthalene group binds with hydrophobic regions of protein. In this study, we analysed the interaction of ANS and MMP-7. At pH 4.5-9.5, ANS inhibited MMP-7 activity in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2). The inhibition was a non-competitive manner and depended on the time for pre-incubation of ANS and MMP-7. At pH 4.5-9.5, the fluorescence of ANS was not changed by the addition of MMP-7. At pH 3.5, MMP-7 lacked activity, and the fluorescence of ANS was increased by the addition of MMP-7. These results suggest that at pH 4.5-9.5, the sulphonic group of ANS binds with MMP-7 through electrostatic interaction, whereas at pH 3.5, the anilinonaphthalene group of ANS binds with MMP-7 through hydrophobic interaction.

Sulfated glycosaminoglycans and sulfated lipids are involved in the biological functions of human matrix metalloproteinase 7 (MMP-7). In this study, the effects of heparin and cholesterol sulfate (CS) on the activity and stability of MMP-7 in the hydrolysis of a synthetic substrate, (7-methoxycoumarin-4-yl)were examined. Heparin increased activity by decreasing K m , and the K m values for 0 and 50 μM heparin were 57 ± 8 and 19 ± 5 μM, respectively. CS decreased activity in a non-competitive inhibitory manner with a K i value of 11 ± 3 μM. In thermal incubation at 50−70°C, heparin increased relative activity (the ratio of k cat /K m of MMP-7 with incubation to that without it), while CS decreased relative activity. These results indicate that heparin increases the activity and stability of MMP-7, while CS decreases them.

Neutral salts activate and stabilize thermolysin. In this study, to explore the mechanism, we analyzed the interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and thermolysin by ANS fluorescence. At pH 7.5, the fluorescence of ANS increased and blueshifted with increasing concentrations (0-2.0 μM) of thermolysin, indicating that the anilinonaphthalene group of ANS binds with thermolysin through hydrophobic interaction. ANS did not alter thermolysin activity. The dissociation constants (K d ) of the complex between ANS and thermolysin was 33 ± 2 μM at 0 M NaCl at pH 7.5, decreased with increasing NaCl concentrations, and reached 9 ± 3 μM at 4 M NaCl. The K d values were not varied (31−34 μM) in a pH range of 5.5−8.5. This suggests that at high NaCl concentrations, Na + and/or Cl -ions bind with thermolysin and affect the binding of ANS with thermolysin. Our results also suggest that the activation and stabilization of thermolysin by NaCl are partially brought about by the binding of Na + and/or Cl -ions with thermolysin.