Physicochemical properties of native and recombinant mungbean (Vigna radiata L. Wilczek) 8S globulins and the effects of the N-linked glycans.
We have previously cloned and characterized the cDNAs of three isoforms of the 8S globulin of mungbean, expressed the major 8Salpha isoform in Escherichia coli, and purified and successfully crystallized it (Bernardo, A. E. N.; Garcia, R. N.; Adachi, M.; Angeles, J. G. C.; Kaga, A; Ishimoto, M.; Utsumi, S.; Tecson-Mendoza, E. M. J. Agric. Food Chem. 2004, 52, 2552-2560). Herein, we report the physicochemical and emulsifying properties of the native 8S and recombinant 8Salpha globulin or vicilin. The circular dichroism spectra analysis of the native 8S and recombinant 8Salpha globulins revealed that the recombinant 8Salpha formed a secondary structure close to that of the native 8S. Further, gel filtration analysis showed that 8Salpha was able to assemble into trimers. The native 8S and recombinant 8Salpha globulins were soluble at pH 3.4 and at pH 7.4-9.0 at low ionic strength, mu = 0.08. Interestingly, the native 8S was more soluble at pH 7.0 and pH 7.4 than the recombinant 8Salpha at mu = 0.08. Both forms were very soluble at pH 3.4-9.0 at high ionic strength, mu = 0.50. The native form exhibited a higher T(m) (69.2, 79.5, and 83.8 degrees C) than the recombinant form (65.6, 71.6, 77.5 degrees C) at mu = 0.1, 0.2, and 0.5, respectively. The recombinant form was found to have greater surface hydrophobicity than the native form. There was little difference in the emulsifying ability between the native 8S and 8Salpha at pH 3.4 and pH 7.6. The results indicate that the presence of N-linked glycans is not essential in the assembly and stable conformation of the mungbean vicilin. However, the N-linked glycans might have contributed to the higher solubility at low ionic strength, greater thermal stability, and decreased surface hydrophobicity of the native vicilin as compared to the recombinant 8Salpha. On the other hand, the N-linked glycans showed little effect on the emulsifying ability of the protein.
Garcia
Roberta N
RN
Institute of Plant Breeding, College of Agriculture, University of the Philippines, Los Baños College, Laguna. rngarciaipb@yahoo.com
Adachi
Motoyasu
M
Tecson-Mendoza
Evelyn Mae
EM
Bernardo
Amy Emiliana N
AE
Utsumi
Shigeru
S
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004355:Drug Stability; D045703:Emulsifying Agents / Q000737:chemistry; D007887:Fabaceae / Q000737:chemistry*; D005916:Globulins / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009994:Osmolar Concentration; D010940:Plant Proteins / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry*; D012995:Solubility
D045703:Emulsifying Agents; D005916:Globulins; D010940:Plant Proteins; D011134:Polysaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins