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. 2012;7(11):e49164.
doi: 10.1371/journal.pone.0049164. Epub 2012 Nov 7.

What are the proteolytic enzymes of honey and what they do tell us? A fingerprint analysis by 2-D zymography of unifloral honeys

Rocco Rossano  1 , Marilena Larocca, Teresa Polito, Anna Maria Perna, Maria Carmela Padula, Giuseppe Martelli, Paolo Riccio
Affiliations

What are the proteolytic enzymes of honey and what they do tell us? A fingerprint analysis by 2-D zymography of unifloral honeys

Rocco Rossano et al. PLoS One. 2012.

Abstract

Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 2-D zymography (2-DZ) of the proteolytic activities present in honeyMT1 extracts.
Proteolytic activities were detected by 2-D zymography on gels copolymerized with gelatin. Gels: A (orange honey), B (eucalyptus honey), C (chestnut honey) and D (sulla honey). 80 µg of proteins were applied to each gel.
Figure 2
Figure 2. 2-D zymography (2-DZ) of the proteolytic activities present in honeyMT2 extracts.
Proteolytic activities were detected by 2-D zymography on gels copolymerized with gelatin. Gels: A (orange honey), B (eucalyptus honey), C (chestnut honey) and D (sulla honey). 80 µg of proteins were applied to each gel.
Figure 3
Figure 3. 2-D zymography (2-DZ) of the proteolytic activities present in honeyPZ1 extracts.
Proteolytic activities were detected by 2-D zymography on gels copolymerized with gelatin. Gels: A (orange honey), B (eucalyptus honey), C (chestnut honey) and D (sulla honey). 80 µg of proteins were applied to each gel.
Figure 4
Figure 4. 2-D zymography (2-DZ) of the proteolytic activities present in honeyPZ2 extracts.
Proteolytic activities were detected by 2-D zymography on gels copolymerized with gelatin. Gels: A (orange honey), B (eucalyptus honey), C (chestnut honey) and D (sulla honey). 80 µg of proteins were applied to each gel.
Figure 5
Figure 5. Specific inhibition by PMSF of the proteolytic activities present in honeyMT1 extracts.
2-D zymography of the honey extracts, in the absence (control, left panels) and in the presence (right panels) of 1.5 mM PMSF, inhibitor of serine proteases. Gels: 1 (orange), 2 (eucalyptus), 3 (chestnut) and 4 (sulla).Only the regions of the gels with isoelectric point between pH 3.5–9.5 and molecular mass between 17–25 kDa are shown.
Figure 6
Figure 6. Bidimensional electrophoresis (2-DE) of proteins present in different unifloral honeyMT1 samples.
2-DE analysis (IEF: linear pH gradient of 3–10; SDS-PAGE: 10% polyacrylamide). Coomassie stained gels: A (orange), B (eucalyptus), C (chestnut) and D (sulla). 300 µg of proteins were applied to each gel. Numbers corresponds to the protein spots identified by MALDI-ToF MS analysis.
Figure 7
Figure 7. Bidimensional electrophoresis (2-DE) of proteins present in different unifloral honeyMT1 samples.
2-DE analysis (IEF: linear pH gradient of 3–10; SDS-PAGE: 15% polyacrylamide). Silver stained gels: A (orange), B (eucalyptus), C (chestnut) and D (sulla). Only the regions of the gels with isoelectric point between pH 4–9 and apparent molecular mass between 14–30 kDa are shown. 60 µg of proteins were applied to each gel. Numbers corresponds to the protein spots identified by MALDI-ToF MS analysis.
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References

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