271058 Confinement Protection Effects of Mesoporous Silica SBA-15 On Myoglobin, in an Environment Inspired by the Groel/ES Chaperonin System

Monday, October 29, 2012: 2:15 PM
Pennsylvania East (Westin )
Michele Lynch1, Justin Siefker2 and Marc-Olivier Coppens1, (1)Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, (2)Dept. Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY

With the increasing prevalence of protein therapy drugs, such as antibodies, it is important to stabilize and protect these proteins in the body. The structure of proteins is more delicate than that of small-molecule drugs; e.g., many proteins denature and become inactive when exposed to proteases or extreme pH [1]. Microscopic, mesoporous silica SBA-15 particles are interesting candidates for drug delivery and sensing applications. SBA-15 contains a hexagonally ordered array of cylindrical pores. These pores have certain commonalities with the GroEL/ES complex, a pair of chaperone proteins found in bacteria cells that assist in the folding of new proteins [3]. More in particular, the pores of SBA-15 have a hydrophilic core, similar to the GroEL/ES complex, and exhibit similar electrostatics with respect to protein adsorption. We report on the denaturation protection from exposure to proteases and extreme pH, conferred to proteins adsorbed on the interior pore surface of SBA-15.

To test for the protective effects of SBA-15, myoglobin is adsorbed on the internal pore surface of SBA-15 particles, and these composites are exposed to the protease pepsin. Both free myoglobin and myoglobin/SBA-15 composites are suspended in phosphate buffer with a pH of 4.6. This pH was chosen because pepsin is only active in environments with a pH lower than 6.5, and optimally at a pH of 2.0 [2]. The activity of free and confined myoglobin is tested via a peroxidase enzyme activity assay, which is measured by UV-Vis spectroscopy. Activity measurements are normalized to those of free myoglobin in buffer with a pH of 7.2 (myoglobin’s isoelectric point), and to the measured amount of adsorbed myoglobin. The myoglobin loading of all composites is below 50 mg/g SBA-15, this is within the Henry adsorption regime.

Our previous work had shown that adsorbing myoglobin to SBA-15 at its isoelectric point (pH 7.2) leads to an extraordinary increase in activity, around 700% [1]. However, at pH 4.6, the activity of confined myoglobin is reduced with respect to that of free myoglobin at the same pH. When free myoglobin is digested by active pepsin, its activity is reduced substantially. In contrast, confined myoglobin shows a nearly imperceptible drop in activity when exposed to pepsin. This result is encouraging, as it suggests that confinement protects the myoglobin from proteolysis.

Changes in secondary structure are observed with Fourier transform infrared spectroscopy (FTIR). Also the effect on myoglobin catalytic activity of restoring the pH of all samples to 7.2 after a temporary decrease in pH will be reported. This experiment is inspired by the behavior of the GroEL/ES complex, which can also refold denatured proteins. The results of this study elucidate the effects of confinement in SBA-15 on myoglobin with respect to extreme pH exposure.

[1] L.C. Sang and M.-O. Coppens (2011), Phys. Chem. Chem. Phys. 13, 6689.

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