Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A.
In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases; D002417:Cattle; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors*; D003902:Detergents / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D000596:Amino Acids; D003902:Detergents; D006146:Guanidines; D012967:Sodium Dodecyl Sulfate; D003851:Deoxyribonucleases; D002268:Carboxypeptidases