1. 1.|We developed a turbidimetric assay system for quantitation of heat-induced protein aggregation which is presumably caused by protein denaturation.
2. 2.|Rhodanese in 6 M guanidinium chloride was employed in the assay system, because this protein recognizes hydrophobic sites on denatured proteins and aggregates.
3. 3.|Turbidity caused by protein-rhodanase aggregation was recorded at 320 nm by using a u.v./VIS spectrophotometer.
4. 4.|When heated, alcohol dehydrogenase (ADH) aggregates with rhodanese. The increase of ADH-rhodanese aggregation was correlated with the loss of enzymatic activity.
5. 5.|These results indicated that the aggregation was proportional to the extent of ADH denaturation which assumingly caused the loss of ADH activity during heating at 45.5°C.
6. 6.|Similar results were observed when cytosolic proteins from CHO cells were heated at 45.5°C. Heated cytosolic proteins promoted aggregation by complex formation with rhodanese. The aggregation increased with increasing heat dose.
7. 7.|Therefore, the rhodanese assay system can be employed usefully to quantitate the protein aggregation after heat stress.
Author Keywords: Turbidimetric assay; rhodanese; protein aggregation; hyperthermia
