EFFECT OF GLYCEROL ON PROTEIN AGGREGATION - QUANTITATION OF THERMAL AGGREGATION OF PROTEINS FROM CHO CELLS AND ANALYSIS OF AGGREGATED PROTEINS

1. In an effort to elucidate the mechanism by which glycerol protects cells against heat killing, the effects of glycerol on heat-induced protein aggregation were studied by employing a cell free system containing cytosolic proteins from CHO cells. 2. Heat-induced unfolding and subsequent aggregation of cytosolic proteins were quantified by a novel turbidimetric assay system utilizing rhodanese (RDN) in 6 M guanidinium chloride. The turbidity caused by protein-RDN aggregation showed a correlation to the amount of aggregated proteins. 3. Aggregation of the proteins was suppressed by glycerol present during the heating in concentration up to 15% (w/v). Glycerol suppressed protein aggregation at various heat doses (0-30 min at 45.5-degrees-C) and temperatures (42-45-degrees-C), implying that glycerol stabilizes proteins against thermal denaturation. 4. The aggregated proteins were analyzed by an SDS-PAGE and individual proteins were quantified. 5. An unknown 28 kDa protein and a 70 kDa protein showed the most pronounced change in aggregation after 30 min heating at 45.5-degrees-C. The 28 kDa protein showed a 5.7-fold increase in control, but only 1.9-fold increase in 10% glycerol. The 70 kDa protein showed a 6.8-fold increase in control, while a 3.5-fold increase in 10% glycerol. 6. Other proteins such as 24, 33, 43, 46, 50, and 55 kDa protein showed 28, 38, 55, 39, 30, and 1 % respective reduction in aggregation by 10% glycerol. 7. These results demonstrate that glycerol affords a differential heat protection for each protein.