FIGURE 6.

Determination of protein stability by limited trypsinolysis. A, thermal stability. 1st lane, 2 μg of purified POR protein; 2nd and 8th lanes, molecular mass markers; 3rd to 7th lanes, 0, 10, 60, 120, and 180 min of trypsinolysis, respectively. Left panels are WT protein, and right panels are A287P protein. Upper panels are reactions performed at 30 °C, and lower panels are reactions performed at 37 °C. The a, b, and c labels in the lower right panel correspond to the bands of 20, 17, and 14 kDa, respectively, as discussed in the text. B, stability with 2′-AMP. 1st lane, 2 μg of protein; 2nd and 8th lanes, molecular mass markers; 3rd to 7th lanes, 0, 10, 60, 120, and 180 min of trypsinolysis, respectively. Left panels are WT protein, and right panels are A287P protein. Upper panels are reactions without 2′-AMP, and lower panels are reactions with 5 mm 2′-AMP. Reactions were performed at 37 °C. C, stability with DLPC. 1st lane, 2 μg of protein; 2nd and 8th lanes; molecular mass markers, 3rd to 7th lanes, 0, 10, 60, 120, and 180 min of trypsinolysis, respectively. Left panels are WT protein, and right panels are A287P protein. Upper panels are reactions performed without DLPC, and lower panels are reactions performed in the presence of DLPC. Reactions were performed at 37 °C. Trypsinolysis was performed as described under “Experimental Procedures.” Trysinolysis experiments (A) were performed three times, with the same results; figure shown is representative.