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Kinetic data measured from folding of the protein interleukin-1β fits best to three exponential phases when studied with tryptophan fluorescence but only two exponential phases when measured using other methods. The technique of ANS fluorescence was used to determine whether the additional phase observed in tryptophan fluorescence was also detected with ANS dye binding. Unlike trytophan fluorescence, the ANS fluorescence was highly dependent on the concentration of protein present during the folding experiment. Experimental controls provide evidence that ANS binds to protein aggregates, present at higher concentrations and absent at lower concentrations. Protein concentration-dependent folding studies demonstrate that, at lower interleukin-1β concentrations, tryptophan fluorescence kinetics can be fit adequately with a two exponential fit. This study indicates that (1) measured interleukin-1β folding kinetics fit to a 2 phase model and (2) at higher protein concentrations, transient association of IL-1β may result in a kinetic fit of 3 phases.