Sodium trichloroacetate as a denaturation reagent for proteins.

Abstract

Trichloroacetic acid (TCA) is known as a precipitant for proteins at acidic pH was applied at neutral pH to proteins such as α-chymotrypsin [EC 3. 4. 4. 5], chymotrypsinogen, lysozyme [EC 3. 2.1.17], ribonuclease A [EC 2. 7. 7.16] and insulin in order to examine its effects on the structures and activities of these enzymes and proteins at neutral pH and to compare them with the effects of urea, guanidine-HCl and halogen derivatives of acetate. The proteolytic activity of α-chymotrypsin was inhibited completely by addition of 1.2 M Na-TCA at pH 7.8, while the activity was fully retained in the presence of 1.5 M sodium monochloroacetate, sodium dichloroacetate, sodium trifluoroacetate, urea or guanidine-HCl at the same pH value. The activities of ribonuclease A and lysozyme near neutral pH were also lost completely with Na-TCA at 1.2M. The four tyrosine residues in the α-chymotrypsin molecule ionized with 0.6M Na-TCA at pH 12.0 while, in the presence of other reagents at 1.5 M, only two or three of them ionized at the same pH value. Some tyrosine residues in chymotrypsinogen, lysozyme and ribonuclease A, which do not ionize at pH 12.0 without reagent, ionized on addition of 1.5 M Na-TCA at the alkaline pH. These proteins when treated with Na-TCA at pH 7.0 underwent spectral shifts due to exposure of tyrosine and/or tryptophan residues from the interior of protein molecules. The helical contents of ribonuclease A and lysozyme increased on addition of Na-TCA, whereas the helical content of insulin and α-chymotrypsin decreased on the same treatment. Na-TCA was thus found to act as a denaturation reagent for proteins which is more effective at neutral pH than the other notable denaturation reagents.