Evaluation of antioxidants in protein formulation against oxidative stress using various biophysical methods.
To evaluate the biophysical stability of protein against oxidative stress, hydrogen peroxide (H2O2) was used to induce non-site-specific protein oxidation. Various biophysical methods were utilized including RP-HPLC, DSC, DLS, and CD. Lysozyme was chosen as a model protein and three different antioxidants (ascorbic acid, N-acetyl-l-cysteine, and l-methionine) were selected to observe their effect. Significant increase in hydrodynamic size, decrease in α-helix propensity, and increase in β-sheet content evident with increasing H2O2 concentration and temperature suggested methionine residues as the most probable site of oxidation. Among the three anti-oxidants, methionine proved superior in suppressing protein oxidation with its increasing concentration. Methionine reacted with H2O2 to form methionine sulfoxide, which aided in decreasing the oxidant concentration to react with the protein. The hydrodynamic size of methionine containing protein was retained when incubated at 40°C after 14 days with unchanged transition temperature (Tm). In contrast, RP-HPLC revealed oxidation alterations when the same samples were stored at 40°C, highlighting the significant impact of temperature on kinetics. N-acetyl-l-cysteine and ascorbic acid were relatively less protective. Their hydrodynamic size was increased with decreasing Tm compared to the reference. In summary, methionine was a superior antioxidant, implicating a promising component in the protein formulation for suppressing oxidation.
College of Pharmacy, Dongguk University-Seoul, Gyeonggi, 410-820, Republic of Korea.
Shavron
S
Hada
College of Pharmacy, Dongguk University-Seoul, Gyeonggi, 410-820, Republic of Korea.
Nam Ah
NA
Kim
College of Pharmacy, Dongguk University-Seoul, Gyeonggi, 410-820, Republic of Korea.
Dae Gon
DG
Lim
College of Pharmacy, Dongguk University-Seoul, Gyeonggi, 410-820, Republic of Korea.
Jun Yeul
JY
Lim
College of Pharmacy, Dongguk University-Seoul, Gyeonggi, 410-820, Republic of Korea.
Ki Hyun
KH
Kim
College of Pharmacy, Dongguk University-Seoul, Gyeonggi, 410-820, Republic of Korea.
Pratik
P
Adhikary
College of Pharmacy, Dongguk University-Seoul, Gyeonggi, 410-820, Republic of Korea. Electronic address: shjeong@dongguk.edu.
Seong Hoon
SH
Jeong
D000818:Animals; D000975:Antioxidants / Q000737:chemistry* / Q000494:pharmacology*; D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004527:Egg Proteins / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D018384:Oxidative Stress / Q000187:drug effects*; D010316:Particle Size; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
D000975:Antioxidants; D004527:Egg Proteins; D011506:Proteins; D009113:Muramidase
Antioxidants; CD; DLS; DSC; Methionine; Protein oxidation; RP-HPLC