Determination of the rotational dynamics and pH dependence of the hydrogen exchange rates of the arginine guanidino group using NMR spectroscopy.

The dynamic behaviour of the guanidino group of arginine has been investigated quantitatively with the intention of providing a set of basis values for the interpretation of data acquired for arginine residues in proteins. At room temperature, a single broad resonance line is observed for the four η-NH(2) protons. Upon cooling the sample (≈10°C at 500 MHz), two η-NH(2) proton resonances are resolved which were shown by HMQC spectroscopy to be the result of slowed rotation about the N(ε)-C(ζ) partial double bond. The flip rate (k(NC)) about the N(ε)-C(ζ) bond was measured as a function of temperature using line-shape analysis of both (1)H and (15)N NMR spectra; at 25°C, k(NC) is between 900 and 1000 s(-1). The exchange broadening, due to N(ε)-C(ζ) bond flips, typically results in weak or missing signals for the η-NH(2) protons of arginine residues in HMQC or INEPT experiments recorded at room temperature, unless the motion is restricted in some way. In a related series of experiments, the pH dependence of the hydrogen exchange rates of the ε-NH and η-NH(2) protons of arginine was measured using saturation transfer (1)H NMR spectroscopy and compared with the equivalent NH(2) protons of the guanidinium ion. As expected, OH ion catalysis dominates over most of the pH range and proceeds at a rate close to the diffusion limit for both types of proton (k(OH)=2×10(9)-1×10(10) M(-1)s(-1), depending on conditions). At low pH values, however, catalysis by H(3)O(+) becomes important and leads to characteristic rate minima in the exchange versus pH profiles. Acid catalysis is significantly more effective for the η-NH(2) protons than for the ε-NH proton; at low ionic strength (50 mM KCl) the rate minima occurred at pH 3.6 and 2.3, respectively. Under these conditions, acid-catalysed rate constants (k(H)) of 706 M(-1)s(-1) (η-NH(2)) and 3 M(-1)s(-1) (ε-NH) were obtained at 25°C. At high ionic strength (1 M KCl) the rate of OH(-) ion catalysis is decreased slightly, whereas the H(3)O(+)-catalysed rate is unchanged. The k(OH) value of the free guanidinium ion is identical to that of the η-NH(2) protons but acid catalysis occurs less easily, leading to a rate minimum at pH 3.3.