Elsevier

Journal of Chromatography A

Volume 1230, 23 March 2012, Pages 48-53
Journal of Chromatography A

Separation of alcohol soluble sorghum proteins using non-porous cation-exchange columns

https://doi.org/10.1016/j.chroma.2012.01.063Get rights and content

Abstract

Kafirins, the storage proteins and major protein of the cereal grain sorghum, play an important nutritional role for millions of people in parts of Africa and Asia. Kafirins are non-water soluble, being soluble only in the presence of detergents or aqueous alcohol mixtures and are among the most hydrophobic of the cereal proteins. Limited Mw heterogeneity of kafirins reduces their resolution when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Charge based separation techniques have been shown to have improved resolution of kafirins, but due to the nature of their solubility, ion-exchange (IE)-HPLC has not been widely used to separate these proteins. To overcome issues of solubility, two different mobile phases were evaluated. The first mobile phase was based on 60% acetonitrile at acidic pH using guanidine-hydrochloride (Gdn-HCl) gradients to elute the proteins from a non-porous cation-exchange column. The second mobile phase tested consisted of 60% acetonitrile using an increasing concentration gradient of a triethylamine phosphate (TEAP) buffer at pH 3.0. The type of alkylation reagent used to stabilize kafirin extracts prior to analysis was found to have an impact on the IE-HPLC separations with the reagent 4-vinylpyridine providing the best resolution. Separations of kafirins in the TEAP mobile phase system resulted in 10 major peaks being resolved. Combining IE-HPLC with reverse phase (RP)-HPLC into 2D separations revealed that the α-kafirins clustered into three major groups not readily apparent in either 1D separation.

Highlights

► We optimized a method for analyzing non-water soluble sorghum proteins by ion-exchange HPLC. ► Ion-exchange separated kafirins with high resolution. ► Combining ion-exchange HPLC with RP-HPLC resulted in two-dimensional separations of kafirins.

Introduction

Cereal grains are a major source of protein for humans worldwide and play important roles in animal feeds. Grain proteins also have important functional roles in food products; the most widely known example of which is wheat gluten. Sorghum grain proteins are no exception and play an important role in the utilization of sorghum and its nutritional properties. The most heavily researched topic with regards to sorghum proteins has been the issue of protein digestibility in both raw flour and cooked sorghum products. Research has shown that while protein digestibility in raw sorghum tends to be close to that of other cereals such as maize, digestibility decreases upon cooking while that of other cereal proteins tends to increase [1], [2]. Reduced digestibility of sorghum proteins may in turn influence starch digestibility [3], which impacts sorghum applications in nutrition, human or animal, and biofuel production.

The major proteins in sorghum are the prolamins. Prolamins are characterized by their solubility in aqueous alcohols and high levels of the amino acids proline and glutamine [4]. Kafirins, sorghum prolamins, have been divided into four subclasses: α, β, γ, and δ based on various factors including solubility and molecular weight [5], [6]. Because kafirins subclasses have been partially defined by differences in their Mw, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been a major tool used to study sorghum proteins. SDS-PAGE typically resolves kafirins into 4–5 major bands spanning a relatively narrow apparent molecular weight range of ∼15–28 kDa [6].

While SDS-PAGE has been useful in studying kafirin proteins, compared to other analytical methods, it has low resolution in separating the kafirin subclasses. This is especially true for the α-kafirins, which are 80–84% of the total kafirins [6]. Recently, it has been reported that there are 19 α-kafirins expressed in sorghum [7] all within a very narrow Mw range of ∼2–3 kDa [6], which would limit the ability of SDS-PAGE to fully characterize the α-kafirin family of proteins. What role, if any, the composition and content of these various α-kafirins may have on sorghum nutritional and functional properties are unknown.

Analytical methods that do not rely on Mw to separate proteins may be useful in further study of the kafirins, especially the α-kafirins. In fact, other analytical methods have been used to separate kafirin subclasses with improved resolution including, isoelectric focusing (IEF) [8], reverse-phase high-performance liquid chromatography (RP-HPLC) [8], [9], [10], [11], and free zone capillary electrophoresis (FZCE) [11]. The highest resolution of kafirins has been achieved with IEF and FZCE, which have resolved α-kafirins into numerous peaks/bands [8], [11]. FZCE separates proteins based on differences in charge density while IEF on the basis of differences in isoelectric point, which is related to charge. Thus, techniques that utilize charge differences offer the highest resolution of kafirins.

One charge based separation technique that has not been widely used to separate cereal proteins is ion-exchange high-performance liquid chromatography (IE-HPLC). This is due to properties of prolamins, the first of which is the fact that prolamins are not water soluble [4]. Detergents, chaotropes, or aqueous organic solvent mixtures are required to solubilize cereal prolamins, all of which complicate the use of IE-HPLC. Detergents can add charges to proteins which would alter their chromatographic behavior as well as interfere with protein binding to ion-exchange columns [12], [13]. Despite these challenges, there have been a few successful reports of IE-HPLC separations of cereal prolamins, such as wheat and barley [12]. Most of these methods have used low levels (1–2 M) of urea in the IE-HPLC mobile phases to maintain solubility of the proteins during separation. However, kafirins have been reported to have reduced solubility in urea even at high concentration (8 M) relative to their solubility in organic solvents such as 70% acetonitrile (ACN) or 70% ethanol [11].

Non-ionic detergents have been used to maintain solubility of membrane proteins during ion-exchange separations, though the detergents have been used at lower levels (e.g. 0.05–0.1%) [14], [15] than typically used to solubilize sorghum proteins (generally 1–2%). In any case, the anionic detergent, SDS, has been found to be the most effective at solubilizing sorghum proteins [16] which would not be suitable for use in ion-exchange chromatography. Mixtures of organic solvents can also be problematic due to the low solubility of many salts in organic solvents [13], [17] and salts such as NaCl can precipitate sorghum proteins from aqueous organic solvents such as 70% ethanol [18].

Despite the poor solubility of many salts in organic solvents, there have been successful IE-HPLC separations of proteins under these conditions. For example, NaClO4, which has good solubility in organic solvents, has been used in combination with up to 70% of ACN to separate proteins by cation-exchange chromatography [13]. Triethylamine phosphate (TEAP) buffers have been used in the presence of high levels of organic solvents in RP-HPLC [19], hydrophilic interaction liquid chromatography (HILIC) [20], [21], size-exclusion chromatography (SEC) [22], and ion-exchange chromatography [21]. Guanidine-hydrochloride (Gdn-HCl) also has good solubility in organic solvents [23] and Gdn-HCl gradients have been used to elute wheat proteins during preparative ion-exchange separations [24]. Gdn-HCl also has the advantage of being a strong chaotrope and may help maintain the solubility of non-water soluble proteins during IE-HPLC in the presence of organic solvents.

IE-HPLC has the potential to provide high resolution separations of sorghum proteins on a platform that can easily be adapted for use as a preparative technique. This is a distinct advantage over both FZCE and IEF. Thus, the goals of this project were to evaluate IE-HPLC mobile phase combinations which would maintain the solubility of the kafirins during separation. Secondary goals were to identify the kafirin subclasses in IE-HPLC separations and compare the resolution to RP-HPLC separations of kafirins as well as the potential for two-dimensional IE × RP-HPLC separations.

Section snippets

Samples and chemicals

Sorghum samples used in this study were grown in either Lane County, Kansas in 2008 or Nebraska in 2003. The maize sample was from a collection of cereal grains at the USDA-ARS, Center for Grain and Animal Health Research (Manhattan, KS). All sorghum flour samples were ground with a UDY cyclone mill 181 (UDY Corporation, Fort Collins, CO) using a 0.5 mm screen. The maize sample was ground using a commercial coffee grinder due to its higher lipid content. HPLC grade acetonitrile was purchased

Mobile phase comparisons

To the best of our knowledge there has only been one report of the use of organic solvent mixtures in IE-HPLC separations of cereal proteins. Gliadins, wheat prolamins, were previously separated using IE-HPLC [26] with a mobile phase containing 30–35% ACN and 0–250 mM NaCl at low pH. While the IE-HPLC method reported in Bietz [26] that was successful for wheat proteins would appear promising, this method is not applicable for sorghum (or likely maize) proteins. The method used for wheat proteins

Conclusions

The non-water soluble sorghum storage proteins, kafirins, were successfully separated with high resolution by IE-HPLC on a non-porous cationic column using mobile phases carefully selected to maintain the solubility of the kafirins during separation. A mobile phase consisting of 60% acetonitrile utilizing a gradient of TEAP was found to provide the best separations. The α-kafirins were resolved into numerous peaks and by combining IE-HPLC with RP-HPLC into 2D separations, the α-kafirins were

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