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Abstract

Surfactants are known to stabilize proteins and are often employed as additives in protein formulations. We have developed a method to study the interaction of these formulation additives with proteins by using the partitioning behavior of a spin label. In protein-free formulations, 16-doxyl stearic acid partitions into micelles above the critical micelle concentration (CMC) of the surfactant and gives rise to composite electron paramagnetic resonance (EPR) spectra composed of spectra from "free" label and "rotationally hindered" label. We compute the fraction of micelle-associated label by factor analysis and generate a label partition curve. When protein is added to the formulation, surfactant-protein aggregates form at concentrations below the surfactant's CMC. Partitioning of the label into these aggregates causes the EPR spectrum to reflect hindered rotation of the label at lower surfactant concentrations than in the protein-free solutions. A simple model of label partitioning shows that these partitioning shifts can be correlated to the surfactant:protein binding stoichiometry. We have studied the interactions of various non-ionic surfactants like Brij and Tween with recombinant human growth hormone and recombinant human interferon-gamma and obtained corresponding binding stoichiometries. These binding stoichiometries match those obtained by other techniques. This technique offers a new method for estimating the protein:surfactant binding stoichiometries.

Surface-Induced Denaturation of Proteins during Freezing and its Inhibition by Surfactants

“…Such interactions have been shown to play an important role in the inhibition of agitation-induced aggregation of human growth hormone. 21 However, again, it seems unlikely that the appropriate protein-surfactant complex could be formed with all of the combinations of proteins and surfactants that we have found inhibit denaturation during freeze-thawing.…”

A transient expansion of the native state precedes aggregation of recombinant human interferon-γ

“…Furthermore, the nature of the intermediate state preceding aggregation has been the subject of much investigation. A number of intermediate conformations have been hypothesized, ranging from transiently expanded species within the native-state ensemble to a moltenglobule structure to a fully unfolded molecule (2,3,12,(17)(18)(19). Ultimately, the nature of the intermediate state is key to understanding the aggregation pathway.…”

Effects of gamma radiation on phase behaviour and critical micelle concentration of Triton X-100 aqueous solutions

“…For example, it is well known that nonionic surfactants also bind weakly to proteins. Surfactant-protein interactions are hydrophobic in nature because proteins with more hydrophobicity bind more surfactants (Bam et al [35]). The mechanism of protein-surfactant interactions depends on the nature and concentration of the surfactant in the solution bulk; e.g., the adsorption of insulin on plastic bags is strongly decreased by the addition of Triton X-100 surfactant to the formulation, since the surfactant reduces the protein's available hydrophobic surface by binding at the hydrophobic patches on the surface of the protein [35].…”

“…Surfactant-protein interactions are hydrophobic in nature because proteins with more hydrophobicity bind more surfactants (Bam et al [35]). The mechanism of protein-surfactant interactions depends on the nature and concentration of the surfactant in the solution bulk; e.g., the adsorption of insulin on plastic bags is strongly decreased by the addition of Triton X-100 surfactant to the formulation, since the surfactant reduces the protein's available hydrophobic surface by binding at the hydrophobic patches on the surface of the protein [35]. Therefore, specific studies considering the individual components used in a formulation should be done to ensure that the surfactant structural changes detected in this investigation do not interfere in the surfactant-protein interactions responsible for solubilization.…”