Abstract
Latex of Maclura spinosa has been used by Kollamalayali tribal community of Western Ghats for curdling of milk. In our previous study we have reported the presence of multiple proteases in the latex, among which the serine protease possesses potent role in hemostasis. With a view to further characterize these proteases the current study was taken up. Maclura spinosa latex contains a serine protease which is resolved using molecular size exclusion column chromatography and an anion exchanger resin in a consecutive manner. The specific activity of the enzyme Maclura spinosa latex protease (MSLP) was found to be 56.15 units/mg and recovery to be 2.68% with a fold purity of 0.41. Being a typical serine protease, MSLP is significantly inhibited by PMSF up to 72.97%. The optimum temperature and pH for the enzyme were found to be 50 °C and 8 respectively. Excision wound healing assay in Swiss albino mice using MSLP, showed accelerated wound closure up to 89.35 ± 1.209% compared to 91.938 ± 1.649% shown by positive control. Further PMSF treated MSLP sample did not show considerable wound healing which confirms exclusive involvement of serine proteases. Examination of biochemical markers viz, hydroxy proline content in healing tissue and catalase activity of fatty tissue also ascertain the potential of MSLP in wound healing. Histopathological studies of healing tissue provide confirmatory evidence with dense collagenation of tissue and fibroblast proliferation rendered by MSLP in the respective treated subjects.
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Abbreviations
- MSL:
-
Maclura spinosa latex
- MSLP:
-
Maclura spinosa latex protease
- PMSF:
-
phenyl methyl sulphonyl fluoride
- IAA:
-
iodo acetic acid
- DEAE-sephadex:
-
diethyl-aminoethyl- sephadex
- p-DMAB:
-
para-dimethyl amino benzaldehyde
- TRIS:
-
trisaminomethane
- MM plot:
-
Michealis–Menten plot
- LB plot:
-
Lineweaver–Burk plot
- Vmax:
-
Maximal Velocity
- Km:
-
Michaelis Constant
- SDS-PAGE:
-
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
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Acknowledgements
The authors wish to thank the management of JSS Mahavidyapeetha, JSS Science and Technology University and JSS Research Foundation, Mysuru for their continued support in this endeavor. The authors acknowledge, TEQIP-III (Technical Education Quality Improvement Programme), a World-Bank-assisted project under the aegis of the Government of India for their financial support.
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Kulkarni, V.B., Achar, R.R., Mahadevappa, M. et al. Hemostatically potent small molecular weight serine protease from Maclura spinosa (Roxb. ex Willd.) accelerates healing of subcutaneous dermal wounds in Swiss albino mice. Biologia 75, 139–149 (2020). https://doi.org/10.2478/s11756-019-00322-y
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DOI: https://doi.org/10.2478/s11756-019-00322-y