N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-L-tryptophan.
BACKGROUND
Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products.


METHODS
Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration.


RESULTS
N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present.


CONCLUSIONS
The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations.


CONCLUSIONS
N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan; Pharma Daiwa Yuge Pharmacy, Kumamoto City, Japan.
Yousuke
Y
Kouno
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan.
Makoto
M
Anraku
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan; DDS Research Institute, Sojo University, Kumamoto 860-0082, Japan.
Keishi
K
Yamasaki
Pharma Daiwa Yuge Pharmacy, Kumamoto City, Japan.
Yoshiro
Y
Okayama
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan.
Daisuke
D
Iohara
Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
Yu
Y
Ishima
Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
Toru
T
Maruyama
Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark.
Ulrich
U
Kragh-Hansen
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan; DDS Research Institute, Sojo University, Kumamoto 860-0082, Japan.
Fumitoshi
F
Hirayama
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan; DDS Research Institute, Sojo University, Kumamoto 860-0082, Japan. Electronic address: otagirim@ph.sojo-u.ac.jp.
Masaki
M
Otagiri
D016166:Free Radical Scavengers / Q000737:chemistry*; D006801:Humans; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry; D010084:Oxidation-Reduction; D055668:Photochemical Processes; D055550:Protein Stability; D017382:Reactive Oxygen Species / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry
D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D012709:Serum Albumin; C006392:N-acetyltryptophan; D014364:Tryptophan; C006379:N-acetylmethionine; D008715:Methionine
Antioxidant activity; Human serum albumin; N-Acetyl-l-methionine; N-Acetyl-l-tryptophan; Octanoate; Photo-oxidation