Revisiting Osborne fractions in the light of barley protein classi-fication

Traditionally, solubility of wheat and barley proteins have been classified according to Osborne fractionation. It con-sists of 1) water extraction of albumins, 2) salt extraction of globulins, 3) alcohol extraction of prolamins, and 4) dilute acid or alkali extraction of glutelins. This classification has laid the foundation for categorizing proteins in barley and other grain plants based on their solubility, but unfortunately leads to overlapping protein fractions, where the same protein can be classified in multiple different groups. It is further complicated by the varying presence of denaturing agents in step 4 or reducing agents in steps 3 or 4 during protein extraction. Thus, comparison of results reported from Osborne fractionation from different studies should be interpreted with caution, keeping the detailed information on the extraction conditions in mind. Furthermore, in the light of current proteomics methods, protein classification solely based on Osborne fractionation seems to be outdated, and other classification systems should be employed, addition-ally focusing on protein function, such as e.g. 1) storage proteins, 2) structural and metabolic proteins, and 3) stress-protective proteins.<br/>In the present study, we first investigated the effect of sieving size (&lt;45 μm to &lt;500 μm) on extraction efficiency of the total protein content from barley flour, followed by Osborne fractionation extraction and subsequent 1D SDS-PAGE analysis of the prolamin fraction. For that, we used flour from three different barley varieties/genotypes (Hordeum vul-gare varieties PS3 and Golden Promise, and Hordeum spontaneum-wild barley genotype) at different combinations of reducing or non-reducing conditions at extraction and electrophoresis. The results showed that the Osborne fractiona-tion resulted in somewhat overlapping protein profiles. Furthermore, attempting to separate barley seed proteins by Os-borne fractionation without the presence of reducing agents proved inadequate in the prolamin fraction using SDS-PAGE, as fewer protein could migrate during electrophorese, likely due to an extensive disulfide network making them too large. This shows that the disulphide-bonded proteins in the prolamin fraction from barley impacts yield relative to extraction conditions in steps 3 and 4 and that the important hordein group in barley cannot be categorized from origi-nal Osborne fractionation criteria alone. Furthermore, interpretation of hordein extraction results needs to be interpreted in the light of redox state depending on the reducing agents at specific steps.<br/>