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 eta-NH2 protons. Upon cooling the sample (approximate to 10 degrees C at 500 MHz), two eta-NH2 proton resonances are resolved which were shown by HMQC spectroscopy to be the result of slowed rotation about the N-epsilon-C-zeta partial double bond. The flip rate (k(NC)) about the N-epsilon-C-zeta bond was measured as a function of temperature using line-shape analysis of both H-1 and N-15 NMR spectra; at 25 degrees C, k(NC) is between 900 and 1000 s(-1). The exchange broadening, due to N-epsilon-C-zeta bond flips, typically results in weak or missing signals for the eta-NH2 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 epsilon-NH and eta-NH2 protons of arginine was measured using saturation transfer H-1 NMR spectroscopy and compared with the equivalent NH2 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 x 10(9) - 1 x 10(10) M(-1)s(-1), depending on conditions). At low pH values, however, catalysis by H3O+ becomes important and leads to characteristic rate minima in the exchange versus pH profiles. Acid catalysis is significantly more effective for the eta-NH2 protons than for the epsilon-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) (eta-NH2) and 3 M(-1)s(-1) (epsilon-NH) were obtained at 25 degrees C. At high ionic strength (1 M KCl) the rate of OH- ion catalysis is decreased slightly, whereas the H3O+-catalysed rate is unchanged. The k(OH) value of the free guanidinium ion is identical to that of the eta-NH2 protons but acid catalysis occurs less easily leading to a rate minimum at pH 3.3.