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 |
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Authors: | Yousuke Kouno Makoto Anraku Keishi Yamasaki Yoshiro Okayama Daisuke Iohara Yu Ishima Toru Maruyama Ulrich Kragh-Hansen Fumitoshi Hirayama Masaki Otagiri |
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Affiliation: | 1. Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan;2. DDS Research Institute, Sojo University, Kumamoto 860-0082, Japan;3. Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan;4. Pharma Daiwa Yuge Pharmacy, Kumamoto City, Japan;5. Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark |
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Abstract: | BackgroundSodium 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.MethodsRecombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4 weeks. 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.ResultsN-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.ConclusionThe 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.General significanceN-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin. |
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Keywords: | HSA, human serum albumin Oct, octanoate N-AcMet, N-acetyl-l-methionine N-AcTrp, N-acetyl-l-tryptophan DSC, differential scanning calorimetry CD, circular dichroism |
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