Conformation of Napin (Brassica juncea) in Salts and Monohydric Alcohols:  Contribution of Electrostatic and Hydrophobic Interactions

Abstract

Napin from mustard (Brassica juncea L.) is a seed storage protein consisting of two subunits linked through disulfide bonds and is predominantly helical in nature. Resistance to trypsin digestion and allergenicity limit its food applications. The role of disulfide linkages, electrostatic as well as hydrophobic interactions, in napin stability have been investigated through spectroscopic methods, employing different fluorescent probes and additives. The subunits are hydrophilic in nature and possess extended structure. With the addition of 0.5 M NaCl, the surface hydrophobicity of napin decreases, whereas the helical content increases by 25%. In the presence of NaCl, emission maximum shifts toward shorter wavelength and the Stern-Volmer constant decreases from 6.5 to 3.4 M-1, indicating compaction of napin. Na2SO4 has no significant effect on the structure due to the lack of a hydrophobic core. In the presence of monohydric alcohols and trifluoroethanol, there is an increase in ordered structure. These studies indicate that the structure of napin, which is hydrophilic in nature, is stabilized by electrostatic interactions, in addition to disulfide linkages.

Effect of pH and NaCl on the Emulsifying Properties of a Napin Protein Isolate

“…It was presumed that the addition of NaCl content caused fluctuations to occur in the NPI conformation leading to slight changes in surface hydrophobicity. Jyothi and others reported a low binding constant of various extrinsic fluorescence probes including ANS (~0.5 mol of probes binding to 1 mol of protein), hydrophilic nature of napin [33]. However in contrast to the present study, Jyothi and others reported a decline in the hydrophobicity of napin with the addition of 500 mM NaCl [33].…”

“…Jyothi and others reported a low binding constant of various extrinsic fluorescence probes including ANS (~0.5 mol of probes binding to 1 mol of protein), hydrophilic nature of napin [33]. However in contrast to the present study, Jyothi and others reported a decline in the hydrophobicity of napin with the addition of 500 mM NaCl [33]. The authors proposed that the high concentration of NaCl lead to the stabilization of a more compacted NPI molecule in solution.…”

“…Protein with good solubility is often associated with high surface charge and low hydrophobicity [31]. Napin is known to have very basic pI (>10.0) and is hydrophilic in nature [17,33]. Although the NPI in the present study exhibited low surface charge, NPI used in this study showed a relatively low hydrophobicity value, which may be the main factor for its excellent solubility across the tested pH levels.…”

“…The zeta potential behavior of NPI in the present study between pHs 3.0 and 4.0 is thus presumed to due to structural changes in the NPI molecule. Jyothi, Singh and Appu Rao showed that with the addition of 0.5 M NaCl, the napin became more compact indicating that there might be changes to the level of exposed amino acid groups on the surface that could lead to changes of surface charge [33]. The addition of NaCl to the NPI solutions resulted in a gradual and steady change in zeta potential as pH declined from pH 8.0 to pH 2.0.…”

Canola/rapeseed protein-functionality and nutrition

“…1) is exhibited as different solubility behaviour compared to cruciferin. Studies of napin of B. juncea (Jyothi et al, 2007) showed that hydrophilic nature of the molecule and the absence of hydrophobic core also influence solubility behaviour of napin while salts such as NaCl tend to stabilize napin structure by compacting. Wanasundara and McIntosh (2013) have reported that not all but a fraction of napin of B. napus and S. alba are soluble in ethanol (70-80%, v/v) and iso-propyl alcohol (30%, v/v).…”