Effect of Arginine on Protein Aggregation Studied by Fluorescence Correlation Spectroscopy and Other Biophysical Methods

Abstract

Arginine has been used extensively as an excipient in the formulation development of protein-based biopharmaceuticals. We investigate the role of arginine in suppressing protein aggregation and its mechanism by using bovine serum albumin as a model system. By using sedimentation velocity and other analytical techniques, we show that the use of arginine inhibits temperature-induced aggregation of the protein. We use fluorescence correlation spectroscopy and other spectroscopic techniques to show that arginine inhibits accumulation of partially folded intermediates, potentially involved in the aggregation process. The hydrodynamic radii of the protein in its native, unfolded, and intermediate states have been determined using fluorescence correlation spectroscopy at single-molecule resolution. A possible mechanism of the effects of arginine and its role as an aggregation suppressor has been discussed.

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics

“…We speculate that alteration of the structure of the hydration layer may be predominately responsible for the formation of these two intermediates with possible minor change in the conformation of the aromatic region of the protein (53). It has been shown recently that arginine may influence the aromatic region of different proteins, although its affinity is weak (31). The absence of any significantly large change in the tryptophan fluorescence or near UV CD justifies the inference of weak interaction.…”

“…1b) whose rate is faster than its molecular diffusion (32, 38 -40). Determination of molecular diffusion ( D ) using a suitable correlation function (Equation 1 for example) yields r H (31,32) (Fig. 1a), which provides information about the conformation of the folded, unfolded, or intermediate states of a protein.…”

“…Labeling of cytochrome c with TMR was carried out using a published procedure (31). Excess free dye was removed by extensive dialysis followed by column chromatography using a Sephadex G25 column equilibrated with 20 mM sodium phosphate buffer, pH 7.5.…”

Interconnection of Salt-induced Hydrophobic Compaction and Secondary Structure Formation Depends on Solution Conditions

“…FCS experiments were carried out with a ConfoCor 3 LSM setup (Carl Zeiss, Evotec, Jena, Germany) using a 40ϫ water immersion objective. The detailed experimental procedure has been published previously (15,16).…”

“…This is a model-free method in which the multicomponent correlation function can be represented by n, the number of non-interacting fluorescent species. These species can have diffusion time values between 0.001 and 500 ms. MEM minimizes the parameter 2 and maximizes the entropic quantity, S ϭ ⌺ i p i lnp i (where p i ϭ a i ⌺ i a j ) to obtain an optimized fit (15,18).…”