Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14-38lAh,, at pH 2.5. These were prepared by chemical synthesis.[RIAbu is a model for reduced BPTI; all cysteine residues are replaced by a-amino-n-butyric acid (Abu). [14-38lAb, retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of Do-the diffusion coefficient extrapolated to infinite dilution-is determined. From Do, a value of the hydrodynamic radius, R,,, is computed from the Stokes-Einstein relationship. At pH 4.5, [RIAbu has an R,, value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental R,, value is the same as for a random coil. In view of the changes in NMR-detected structure of [RIA,,,, at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 3413974-13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non-native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14-38IAbu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14-38lAh,, relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under "molten globule" conditions. Keywords: bovine pancreatic trypsin inhibitor; protein folding; protein hydrodynamics; pulsed field gradient NMR; unfolded proteinsThe structure of polypeptides at the onset of folding is a fundamental issue in protein folding. Two questions concerning unfolded proteins are of special interest: The tendency of the unfolded chain to form non-random conformations, and the extent of collapse of the polypeptide prior to formation of secondary structure.