Studies on the enzymatic and physicochemical behaviour of the trichloroacetic acid-treated and untreated bovine pancreatic ribonuclease.
Exposure of ribonuclease A to 5% trichloroacetic acid inactivates the enzyme partially. One of the possible reasons for such inactivation might be the exposure of one of the buried tyrosyl groups to the outside surface of the molecule (Sagar and Pandit (1983) Biochim. Biophys. Acta 743, 303-309). The trichloroacetic acid-treated enzyme hydrolysed 2':3'-cCMP with an efficiency of about 60%; while with rRNA as substrate, it is about 45%. Results indicate that apart from the reduction in the activity on trichloroacetic acid treatment, the enzyme possesses a reduced ability to break down the secondary structures of substrates such as rRNA in the first phase of the reaction. Thermal unfolding of ribonuclease A was followed by various physicochemical techniques such as UV absorbance, CD-spectroscopy and differential scanning microcalorimetry. The results indicate that the enzyme, after trichloroacetic acid-treatment, has a less ordered structure when compared to that of untreated enzyme. Thermal unfolding profiles reveal that trichloroacetic acid-treated ribonuclease A, like the untreated enzyme, follows a one-step transition with relatively lower transition temperature (Tm). NMR-spectral data suggests perturbations in the histidyl environment at the active site.
Sagar
A J
AJ
Centre for Cellular and Molecular Biology, Hyderabad, India.
Subbiah
V
V
Pandit
M W
MW
D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D003563:Cyclic CMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006639:Histidine; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011392:Proline; D011487:Protein Conformation; D011489:Protein Denaturation; D012335:RNA, Ribosomal / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013816:Thermodynamics; D014238:Trichloroacetic Acid / Q000494:pharmacology*
D006146:Guanidines; D012335:RNA, Ribosomal; D003563:Cyclic CMP; D006639:Histidine; D014238:Trichloroacetic Acid; D011392:Proline; D012259:Ribonuclease, Pancreatic; D019791:Guanidine