3.1 Electrophoretic profile properties of proteins
The primary and tertiary structure of proteins was examined by SDS-PAGE to estimate molecular weights of polypeptides and their complexes and to compare their respective band intensities.
In 1991the proposed nomenclature of kafirin polypeptides (subunits) was established based on their similarities with those of zein 18. More recent studies have revealed greater hydrophobicity with more dominance of α-helical secondary structure in grain kafirin than zein 5, 19. However, zein is still considered an appropriate polypeptide identification standard for kafirins and was used as an internal control in this study.
A typical electrophoretogram under non reduced conditions of protein marker (lane 1), zein (lane 2) and kafirin from sorghum grain (lane 3) and sorghum DDGS kafirin (lane 4) is given in Fig. 1. For zein, the banding pattern was in line with those previously reported 18, 20, 21. The zein shows a band at ~ 19000 KDa corresponding to β polypeptide monomer, a band at ∼ 21,000–23,000 KDa corresponding α1 and α2 polypeptides. Some dimers were clearly visible in from ∼ 43000–50000 KDa and a few faint oligomer bands at ∼ 63000–67000 KDa.
Kafirin from both sorghum grain and DDGS had very similar banding patterns. Both had a major band at ∼ 21000 to 23000 KDa indicative of α1 and α2 kafirin polypeptides 5. A faint band was seen in both kafirins seen at ∼20000 KDa indicative of β polypeptide monomer. In addition to monomers, a band at ∼ 47000 KDa in both kafirins suggests the presence of dimers. Faint bands at ∼ 67000–86000 KDa are also seen corresponding to oligomers and a distinct band at the bottom of the loading wells indicating presence of highly polymerized kafirins that were not able to enter the gel.
The similar electrophoretic patterns of both grain and the DDGS kafirins indicate that the kafirin polypeptides were stable to the harsh conditions used in the sorghum bioethanol production process (eg. heat and pressure) that resulted in the DDGS.
3.2 Characterising protein secondary structure using attenuated total reflection-Fourier transform IR spectroscopy (ATR-FTIR)
Characterising the secondary structure of proteins (e.g., prevalence of β-sheets, α-helices) is important as it can affects their functionality in related to material behaviour. No differences were visible in FTIR spectrum of the two kafirins. Figure 2A is a representative FTIR spectrum the kafirin, showing the characteristic amide I (νC = O of amide functional group) and amide II (δN-H of amide bond) bands. The amide I band is shaded in pink, the amide II band is that directly to the right. It is well established in the literature that that the amide I band is highly sensitive to the molecular geometry and conformational state of the protein secondary structure because 1) different secondary structures are stabilised by the inherently unique patterns of hydrogen-bonding across the carbonyl group in the amide linkage, 2) the νC = O vibrational frequency of the amide group is altered by hydrogen bonding 22, 23, 24, 25. Therefore, the position and shape of the amide I band serves as a fingerprint of protein secondary structure 23, 26. Although absolute determination of protein secondary structures is difficult from analysis of the amide I band alone, identification of relative changes in protein secondary structure has been routinely undertaken by many, including in previous studies of kafirin 23, 26, 27, 28, 29.
Although appearing as a single broad peak in the raw data (Fig. 2A), the amide I band is actually the result of multiple overlapping absorbance bands, each representing an underlying secondary structure. The position of the underlying bands can be identified through spectral deconvolution approaches, such as calculation of second-derivatives (Fig. 2B), which artificially decrease band-width enhancing spectral resolution. As can be seen in Fig. 2B, the amide I band of both kafirins consist of 9 underlying bands, centred at ~ 1686, 1677, 1669, 1660, 1650, 1643, 1634, 1623, and 1610 cm− 1. These second-derivative spectra indicate a difference in secondary structure between grain kafirin and DDGS kafirin by a greater secondary derivative intensity at 1620 cm− 1 in the latter.
To validate the results of the second-derivative spectra of the kafirins, curve fitting approaches were used to the fit original non-derivatised spectrum to a linear combination of underlying components. Curve fitting was performed using a linear least squares algorithm, and included two additional bands to those identified from second-derivatives, to account for absorbance contribution from the neighbouring ester carbonyl and amide II bands. The relative distribution of underlying components of peaks with major difference as examined on second derivative spectra centred at 1620, 1634, and 1643 cm− 1 of DDGS kafirin compared to grain kafirin is shown in Fig. 2C. These overlapping fits confirm altered protein secondary structure shape between DDGS kafirin and grain kafirin.
The complete curve fitting to determine the area underneath each of 9 underlying bands of amide I of the second derivatives for the two kafirins are represented in Fig. 2D (grain kafirin) and Fig. 2E (DGGS kafirin). A full assignment of each underlying amide I component to a protein secondary structure is beyond the scope of this paper. However, on comparing curve fits, that for DDGS kafirin appears to have higher contribution for the underlying component centred around 1620 cm− 1 and lower contribution from underlying components centred at 1643 cm− 1 and 1634 cm− 1 than grain kafirin. The integrated areas under the curves of amide I as determined by curve fitting Fig. 2D and E) were then statistically analysed (n = 5, students’t-test, 95 % confidence interval) and presented in Fig. 2F.The key finding this study supports increase in area of peak centered at 1620 cm− 1 (***p < 0.0005) but also suggest decrease in area of peak at 1643 cm− 1 (*p < 0.05) and 1634 cm− 1 (**p < 0.005). It has been established extensively in the literature 30 that the underlying component at ~ 1623 cm− 1 likely results from proteins with an aggregated β-sheet secondary structure. The band centred around 1643 cm− 1 is likely to arise from random or disordered secondary structures, and the band at 1634 cm− 1 is likely to arise from non-aggregated β-sheets. There is some ambiguity in these assignments however, as the band at 1643 cm− 1 could also contain contributions from 310Helices, or even from strongly hydrogen bonded α-helices. Nonetheless, regardless of this ambiguity, the results strongly support a higher level of extended β-sheet aggregates and lower levels of α-helix and random coils in DDGS kafirin. Such a finding is consistent with past literature investigating the effect of high temperatures on the protein secondary structure of purified kafirin, which indicate a propensity of the protein to produce aggregated β-sheet structures 28.
We hypothesise that this is because of during the biofuel manufacturing process the high temperature were capable enough to unravel some α-helices and random coils followed by realignment and reorganisation into β-sheets. The higher level of β-sheets in DDGS kafirin compared to grain kafirin may make the former better suited for viscoelastic self-assembly delivery systems. It is well documented in literature that biomaterial with viscoelastic properties have high energy absorption capacity, shock absorbance behaviour and dumping response 31, 32, 33. Thus, it become evident that DDGS kafirin might retain architecture of formulated biomaterial system in physiological environment than that of commonly used elastic biomaterials which are usually thought to be loading dependent only. The DGGS kafirin may endow interfacial wettability to biomaterials because of more aggregated β-sheets, which are believed to be less hydrophobic than α-helix.
3.3 Characterising protein diffraction patterns using X-ray diffraction (XRD)
The XRD patterns of DDGS kafirin, grain kafirin and zein exhibit two broad peaks at approximately 9 and 20° 2θ (Fig. 3A), which is consistent with previous XRD investigations of prolamin proteins 16, 34. In general, diffraction studies are not “information rich” with respect to determining the structure of prolamins, largely due to a high degree of unordered structure within prolamins, which may be amorphous or nano-crystalline 35, 36. The results of this study are consistent with the literature and indicate a low degree of order of nano-crystalline structure in the zein, grain kafirin and DDGS kafirin, which is evident through the large widths of the peaks centred at ~ 9 and 20° 2θ. As the peak centred at 20° 2θ was asymmetric, curve fitting was undertaken. The results indicate that 3 underlying curves (at ~ 19.5, 24, and 30° 2θ, all constrained to have the same peak width) are required to fit the peak at 20° 2θ, in the case of zein and grain kafirin and fits reveal an additional peak is present in DDGS kafirin at 19° 2θ
The Fig. 3B, highlights increased intensity at 20° 2θ, and increased 20° to 9° 2θ area ratio in DDGS kafirin. The impact of this final peak on the XRD patterns can be observed as an altered, more narrow and intense peak shape at 20° 2θ in the pattern of kafirin DDGS. Likewise, the ratio of the peak areas of the peak at ~ 9° 2θ and 20° 2θ is slightly lower in DDGS kafirin than the zein or grain kafirin.
These differences in the diffraction patterns in DDGS kafirin relative to zein and grain kafirin support a difference in protein structure, most likely reorientation 37 and packing of the molecules in to a more ordered arrangement 38 in the DGGS kafirin. As the FTIR spectroscopic data (Sect. 3.2) indicated increased β-sheet aggregates in the DDGS kafirin, which are often fibrillar in nature, an increased in β-sheet fibrils in DDGS kafirin could account for these XRD results.
Another important observation from the XRD data is that although differences in structure between grain kafirin and DDGS kafirin and are apparent, the crystallite size does not appear to have changed substantially (Table 1).
Table 1
Diffraction peak position, crystallite size and relative peak areas for zein, grain kafirin and dried distillers grain with solubles (DGGS) kafirin as measured by cx-ray diffraction (XRD).
Sample | Phase | Peak position (° 2θ) | Crystallite size, Lvol (nm) | Area (net) | Area (rel.) |
Zein | Alpha | 8.87 | 1.1 | 128 | 0.123 |
Beta | 19.6 | 1.1 | 565 | 1 |
24.2 | 324 |
30.0 | 152 |
Grain kafirin | Alpha | 8.87 | 1.0 | 132 | 0.127 |
Beta | 19.7 | 1.1 | 565 | 1 |
24.4 | 332 |
30.4 | 141 |
DDGS kafirin | Alpha | 8.9 | 1.0 | 109 | 0.118 |
Beta | 19.6 | 1.1 | 517 | 1 |
23.8 | 292 |
27.7 | 110 |
Sharp | 19.0 | 5.6 | 19.6 | |
31.6 | 5.0 | 19.7 | |
3.4 Characterising protein in-solution structure using small-angle X-ray scattering (SAXS)
SAXS was used to assess morphological features of zein and the kafirins. The scattering curves were well-fitted with a two-level unified fit model 15 combining Porod and Gunier models, where the first level describes surface scattering from very large particles, and the second level describes scattering from ~ 25 Å particles (Fig. 4).
At low scattering angles, the data can be modelled with a Porod exponent of ~ 3, ~2.4 and ~ 3.5 for zein, grain kafirin and DDGS kafirin, respectively 39. These exponents show that the proteins show properties of surface (3 < P < 4) and volume (P < 3) fractals, showing self-similar behaviour in their structure. If there is no interatomic interaction between molecules, radius of gyration (Rg) can be directly linked with mean square of interatomic distances of molecules present in solution medium 40, allowing the average size of zein and kafirins to be determined. The calculated radii of gyration were 25.8, 24.8, and 28.4 Å for zein, grain kafirin and DDGS kafirin, respectively. These data indicate a difference in the tertiary/quaternary structure of DDGS kafirin, as indicated by the increased radius of gyration, relative to zein and grain kafirin.
3.5 Characterising protein surface morphology and elemental composition using field emission-scanning electron microscopy (FE-SEM) and energy-dispersive X-ray spectroscopy (EDS)
The morphological analysis and surface chemical composition of the proteins were examined by FE-SEM and EDS. Figure 5 (A-C) depicts morphological micrographs and Fig. 5 (D-F) the surface elemental composition of DDGS kafirin, grain kafirin and zein respectively.
The DDGS kafirin shows irregular surfaced large aggregates of shapes with internal pores, Fig. 5A while grain kafirin was more spherical with homogeneous surface, Fig. 5B and zein showed non uniform surface assembling characterises, Fig. 5C. The formation of compact micro spherical shapes with more homogeneity in grain kafirin. Thus, it can be hypothesis that assembling grain kafirin into ordered structure need no external guidance but relies on internal interactions such as vander walls, hydrogen bonding (S-S), capillary and π-π. However solvent in use and size of molecule (η/µ) might be of interest in formation mechanism.
The formation of the larger particles in DDGS kafirin might be related to heat induced transformations in the higher structures during manufacturing of ethanol. This is in agreement with the FTIR data which shows higher abundance of aggregated β-sheets in kafirin DDGS. Heat induced disruption of disulphide bonds (S-S) resulting in re-association of polypeptides into large compact micro aggregates 41, 42, 43 could be a possible pathway to aggregated β-sheet formation. Another possible formation mechanism for the aggregates is that some polypeptides were denatured during the bioethanol production system leading to a subsequent increased propensity to form aggregates.
The elemental profile across the surface of DDGS kafirin, grain kafirin and zein was examined by EDS, and is represented in Fig. 5 (D-F). The EDS showed an abundance of C, N, O and S on the surface of grain kafirin Fig. 5F and zein Fig. 5E. The zein literature suggested formation of surface segments with these elements have high hydrophobicity with contact angle of 126° 17. The presence of Pt in DDGS kafirin and zein is because of the sputter coating.
The surface elemental composition of DDGS kafirin was different than grain kafirin and zein. In addition to C, N, O and S elements some more elements Na, Cl and K were present, Fig. 5D. It is a well-known fact that elements facing bottom left corner of periodic table such as sodium, potassium and etc are more active suggesting increased reactive surface sites for DDGS than grain kafirin and zein.
The novelty of the DDGS kafirin is that despite presence of the additional elements on surface, atomic radius of C and N element on surface remained same and atomic radius of S and O element increased. Although, additional elemental profile gives a clear understanding that DDGS kafirin might endow interfacial wettability. Thought striking, increased atomic radius of elements (S & O), which are proven to form hydrophobic surface sites 17, and additional more reactive elements suggest DDGS surface might have both hydrophobic and hydrophilic surface segments.
Thus, we hypothesise that compared to grain kafirin, DDGS kafirin if used as an active encapsulating agent might have enhanced solubility and formation of complexes with target compounds in aqueous systems. These differences in structure and functionality may also be explained by heat induced transformations in the molecular architecture of DDGS kafirin during the bioethanol production process.