(593ai) Effect of Carbohydrates On the Interaction Between Lysozyme and Procyanidin

The interaction of procyanidins with proteins has aroused extensive attention due to its important relationship with many biologically relevant processes and the astringent property of polyphenols in red wine [Mcrae and Kennedy, 2011]. Moreover, this interaction often leads to the formation of complex aggregates, disruption of multilevel structures of proteins and the loss of biological activity [Goncalves et al., 2010]. Therefore, a deep understanding of the interaction mechanism is critical to control the aggregation behavior and improve the quality of red wine.

This present work investigated the interaction between procyanidin B3 and lysozyme using various biophysical methods. Firstly, we characterized the multilevel structural changes (namely, quaternary, tertiary and secondary) of lysozyme during its interaction with procyanidin B3 by light scattering techniques, fluorescence measurement, and circular dichroism. Then, the effect of two carbohydrates, gum arabic and sucrose, on the aggregation between lysozyme and B3 was also studied inspired by the phenomenon during the fruit ripening stage. The aggregation mechanism and carbohydrate inhibition behavior were analyzed. Moreover, the change of lysozyme activity was correlated with its structural alterations during aggregation. The main ideas and conclusions are summarized as follows.

(1) Light scattering techniques were employed to investigate the formation of lysozyme/procyanidin B3 aggregates. Dynamic light scattering results showed that a dramatic increase in the size of aggregates was initially observed after the addition of procyanidin B3. The average size increased slowly with continuously increasing procyanidin B3 concentration. These results clearly indicated the complex formation produced by the strong binding affinity of procyanidin B3 to lysozyme. Nephelometry technique also confirmed the increased size of the dispersed insoluble aggregates with increasing B3 concentrations

(2) Fluorescence measurements were conducted to analyze the tertiary structure changes of lysozyme. A progressive decrease in the fluorescence intensity was caused with increasing B3 concentrations, which indicated an interaction between lysozyme and B3. The quenching process analysis according to the modified Stern–Volmer equation indicated a static quenching which suggested the ground state complex formation between B3 and lysozyme. Moreover, synchronous fluorescence spectroscopic studies show the maximum emission wavelength of Tyr remained unchanged, whereas the emission maxima of Trp had a faint red shift (1.0 ± 0.1 nm). This observed red shift indicating a change in the microenvironment of Trp from a relatively hydrophobic core to a polar aqueous environment resulted from unfolding of the compact tertiary structure of lysozyme. In addition, the quenching of lysozyme fluorescence may be responsible for the loss of enzyme activity, because the mainly intrinsic fluorescence residues (Trp 62 and Trp 108) are arranged close to the substrate-binding site and play important roles in binding with substrate [Rothwarf and Scheraga, 1996].

(3) Circular dichroism spectroscopy was performed to evaluate the effect of B3 on the secondary structural changes of lysozyme. The gradually increased intensity at 208 nm demonstrated the binding of B3 to lysozyme disrupts the protein secondary structure upon complexation. The α-helical content of lysozyme decreased from 24.66% to 10.9% at a molar ratio of lysozyme to B3 of 1:10. Meanwhile, lysozyme activity against M. lysodeikticus gradually decreased from 4.465×10⁴ U/mg to 3.186×10⁴ U/mg. This result combined with the structural changes of lysozyme during aggregation confirmed that the conformation of a protein determines its biological function.

 (4) Sucrose and gum arabic have the ability to inhibit the formation of insoluble protein/procyanidin aggregates. A rapid decrease in hydrodynamic radius of the aggregates was observed with the addition of gum arabic, while sucrose had a lesser reductive effect on complex formation. Moreover, the interaction of lysozyme/B3 with gum arabic does not restore the fluorescence and activity, combined with the reduction in aggregate size, support the idea that gum arabic partially disassociates lysozyme/B3 complexes and induces the formation of a more soluble ternary complex between lysozyme/B3 and gum arabic [Mateus et al., 2004]. For sucrose, the aggregate size decreases concomitantly with the recovery of fluorescence and enzymatic activity may be explained by the association of the low molecular weight carbohydrates with the polyphenols during incubation. This molecular association in solution prevents polyphenols from further binding to the protein, thus inhibiting aggregate formation. These findings provide deep insights into the mechanism of aggregation and  carbohydrate inhibition between lysozyme and procyanidin B3.

This work was supported by the Natural Science Foundation of China (No. 20806057 and 31071509), the Ministry of Science and Technology of China (Nos. 2012BAD29B05 and 2012AA06A303), and the Ministry of Education (No. NCET-11-0372).

References

1. Mcrae J. M. and Kennedy J. A. Wine and Grape Tannin Interactions with Salivary Proteins and Their Impact on Astringency: A Review of Current Research, Molecules, 16, 2348-2364, 2011
2. Goncalves R., Mateus N. and de Freitas V. Biological Relevance of the Interaction between Procyanidins and Trypsin: A Multitechnique Approach, Journal of Agricultural and Food Chemistry, 58, 11924–11931,2010
3. Rothwarf D. M. and Scheraga H. A. Role of Non-Native Aromatic and Hydrophobic Interactions in the Folding of Hen Egg White Lysozyme, Biochemistry, 35, 13797–13807, 1996
4. Mateus N., Carvalho E., Luis C. and de Freitas V. Influence of the tannin structure on the disruption effect of carbohydrates on protein–tannin aggregates, Analytica Chimica Acta, 513, 135–140, 2004