Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two-dimensional zymography and direct identification of zymographic spots by mass spectrometry.
BACKGROUND
Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow-up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases for meat tenderisation and other industrial purposes.


RESULTS
Kiwi crude extracts were analysed by two-dimensional zymography on gelatin-containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI-ToF mass spectrometry for protein identification. The most representative enzyme was actinidin.


CONCLUSIONS
The innovative achievements of the present study are the: (1) two-dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI-ToF MS analysis of the zymographic spots.
Larocca
Marilena
M
Department of Biology, Defence and Agro-Forestal Biotechnology, Center of Bioproteomics, University of Basilicata, 85100 Potenza, Italy.
Rossano
Rocco
R
Riccio
Paolo
P
D029042:Actinidia / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D010450:Endopeptidases / Q000032:analysis*; D005638:Fruit / Q000201:enzymology*; D006868:Hydrolysis; D010936:Plant Extracts / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010936:Plant Extracts; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C018745:actinidain