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Turbidity Assay for Rapid and Efficient Identification of High Protein Digestibility Sorghum Lines¹

¹Paper 16548 of the Purdue University Agricultural Research Programs. ²Department of Food Science and the Whistler Center for Carbohydrate Research, Purdue University, West Lafayette, IN. ³Department of Agronomy, Purdue University, West Lafayette, IN. ⁴Corresponding author. E-mail: hamakerb@foodsci.purdue.edu.

Recently, our laboratory reported a protein digestibility assay based on SDS-PAGE that distinguishes mutant high protein digestibility from wild-type sorghum lines. Using that assay, high protein digestibility sorghum lines were identified both qualitatively (visual observation) and quantitatively by measuring the SDS-PAGE band intensity of the undigested α-kafirin protein. Here, we report on a new turbidity assay that can be used for an even quicker quantitation of the undigested proteins with much higher throughput for screening purposes. Proteins remaining after 1 hr of pepsin digestion were extracted with a buffer of SDS, 2-mercaptoethanol, and borate and an aliquot of the extract was precipitated using 72% trichloroacetic acid (TCA). Absorbance of the resulting turbid solution was then read at 562 nm. Lower readings corresponded to more digestible lines. The turbidity of the suspensions developed quickly and reached a plateau at ≈5 min for high protein digestibility lines and 10 min for wild-type lines. The turbid solutions remained stable for at least 1 hr. Two distinct groups, wild-type and high protein digestibility sorghum lines, were obtained when the assay was compared with a standard pepsin digestibility procedure and to our recently developed SDS-PAGE assay. A comparison with the bicinchoninic acid (BCA) assay of protein quantitation indicated that the turbidity assay is more efficient in differentiating between wild-type and high protein digestibility sorghum lines. We have further refined the turbidity assay for microtiter plate analysis making it possible for a single operator to analyze ≈200 sorghum lines per day, compared to 60 lines when using the SDS-PAGE assay.