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The interaction of Na(+) and K(+) with proteins is at the basis of numerous processes of biological importance. However, measurement of the kinetic components of the interaction has eluded experimentalists for decades because the rate constants are too fast to resolve with conventional stopped-flow methods. Using a continuous-flow apparatus with a dead time of 50 micro s we have been able to resolve the kinetic rate constants and entire mechanism of Na(+) binding to thrombin, an interaction that is at the basis of the procoagulant and prothrombotic roles of the enzyme in the blood.

In order to understand the mechanism by which a polypeptide chain folds into its functionally active native state it is necessary to characterize in detail all the species accumulated along the pathway. The elusive nature of protein folding intermediates poses their identification and characterization as an extremely difficult task in the protein folding field. In the case of small single domain proteins, the direct measurement of the thermodynamics and structural parameters of protein folding intermediates has provided new insights on the nature of the forces involved in the stabilization of nascent protein structures. Here we summarize some of the experimental approaches aimed at the detection and characterization of folding intermediates along with a discussion of some general structural features emerging from these studies.