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Effects of 8‐Arm Polyethylene Glycol (PEG 8 ) Addition on Thermal Stability of Bovine Serum Albumin (BSA)
Author(s) -
Parker Jacob S,
Hill Jonathan,
Olsen Kenneth W
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.922.12
Subject(s) - bovine serum albumin , chemistry , polyethylene glycol , pegylation , peg ratio , chromatography , sodium dodecyl sulfate , nuclear chemistry , organic chemistry , finance , economics
Bovine Serine Albumin (BSA) conjugated to 8‐arm Polyethylene Glycol (PEG‐‐‐‐ 8 ) via Strain‐Promoted Azide‐Alkyne Cycloaddition (SPAAC) has been proven to be a viable drug delivery system. In our experiments, BSA was de‐fatted and then activated using a short dibenzocyclooctyne (DIBO) as well as an extended DIBO activator (DIBO‐PEG 4 ). The activated BSA was then conjugated to N 3 ‐functionalized PEG 8 and purified via size exclusion chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) of the product showed multiple species verifying the cycloaddition. BSA has two main drug binding sites, DS1 and DS2. It has been shown sulfamethoxazole (SMZ) preferentially binds to DS1 while phenytoin (PHT) preferentially binds to DS2. Fluorimetry was performed using SMZ and PHT to test the functionality of both drug binding sites of PEGylated BSA at room temperature proving both sites functional after PEGylation. The objective of this study is to explore the potential usage of this product by analyzing thermal stability of the PEG 8 ‐BSA x compound. In this study, UV‐Visual Spectroscopy (UV‐Vis) was used to monitor aggregation of denatured PEG 8 ‐BSA x over a range of temperatures. Initial assays via UV‐Vis showed less aggregation of PEGylated sample than unmodified BSA. Circular Dichroism (CD) was then performed to determine the temperature range conjugated BSA would remain functional as indicated by maintenance of its secondary structure. To verify the functionality of PEGylated BSA after exposure to high temperature followed by cooling, fluorimetry experiments were repeated to measure drug binding of SMZ and PHT.