Abstract
Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC 5) and CCACC (dC 2AC 2). This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions. Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations. Samples at equilibrium were infused directly into the mass spectrometer under native conditions. For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC 5 and dC 2AC 2 dissociation constants was problematic.
Highlights
Bovine pancreatic ribonuclease A (RNase A) is an endoribonuclease (EC 3.1.27.5) that hydrolyzes RNA
In the context of an undergraduate lab, desalting would introduce students to a common sample preparation technique. It is unclear why the decrease in the total ion abundance for the ligand-bound forms of RNase A was observed at higher deoxyoligonucleotide concentrations (Figure 2)
The ion intensity ratio of free RNase A to the RNase A+cytidine 2′-monophosphate (2′-CMP) complex was observed to vary with charge state as follows: +8 (0.65), +7 (0.73), +6 (1.1)[12]
Summary
Bovine pancreatic ribonuclease A (RNase A) is an endoribonuclease (EC 3.1.27.5) that hydrolyzes RNA. It is a small single chain polypeptide (124 amino acids) containing four disulfide bridges and is known for its significant stability[1]. RNase A has been called “the most studied enzyme of the 20th century” and it has seen wide use as a model protein in biochemical and biophysical experiments[1]. RNase A is an excellent model for undergraduate lab experiments, because it has been extensively studied, and because its use presents an opportunity to reemphasize important concepts in biochemistry and biology
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