Determination of oligomeric state of BmDHS

The oligomeric state of BmDHS in solution was determined through the application of the purified protein onto the Superdex 200 Increase 10/300 (GE Life Science). Three different runs were used to obtain the standard curve, in which two of them contained a standard mix of proteins and the other one contained blue dextran 2000 to obtain the void volume. Firstly, blue dextran was applied, followed by the first mix with catalase (232 kDa), conoalbumin (75 kDa), and carbonic anhydrase (29 kDa). The second mix was composed by aldolase (158 kDa), ovoalbumin (44 kDa), and Ribonuclease A (13.7 kDa), then, 900 μg of purified BmDHS was applied onto the column. The standard proteins and the blue dextran 2000 belong to the low and high molecular weight gel filtration calibration kits (GE Life Sciences).

The proteins were solubilized in gel filtration buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 5% Glycerol, 0.5 mM TCEP). The AKTA 25 L system flow was set up to 0.4 mL.min^-1 and one milligram of each standard protein were loaded in a 500 μL injection. The elution volumes were used to calculate Kav values of each protein according to the following equation: Kav = (VE-V0) / (VC-V0), where VE, V0, and VC are the elution, void, and column volumes respectively. Kav values were plotted against the log of the molecular mass of the proteins to calculate the curve equation and discover the BmDHS mass in solution.