T-1 relaxation in the rotating frame (T-1rho) is a sensitive magnetic resonance imaging (MRI) contrast for acute brain insults. Biophysical mechanisms affecting T-1rho relaxation rate (R-1rho) and R-1rho dispersion (dependency of R-1rho on the spin-lock field) were studied in protein solutions by varying their chemical environment and pH in native, heat-denatured, and glutaraldehyde (GA) cross-linked samples. Low pH strongly reduced R-1rho in heat-denatured phantoms displaying proton resonances from a number of side-chain chemical groups in high-resolution H-1 NMR, spectra. At pH of 5.5, R-1rho dispersion was completely absent. In contrast, in the GA-treated phantoms with very few NMR visible side chain groups, acidic pH showed virtually no effect on R-1rho, The present data point to a crucial role of proton exchange on R-1rho and R-1rho dispersion in immobilized protein solution mimicking tissue relaxation properties.