Yousuke Kouno1, Makoto Anraku2, Keishi Yamasaki3, Yoshiro Okayama4, Daisuke Iohara2, Yu Ishima5, Toru Maruyama5, Ulrich Kragh-Hansen6, Fumitoshi Hirayama3, Masaki Otagiri7. 1. Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan; Pharma Daiwa Yuge Pharmacy, Kumamoto City, Japan. 2. Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan. 3. Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan; DDS Research Institute, Sojo University, Kumamoto 860-0082, Japan. 4. Pharma Daiwa Yuge Pharmacy, Kumamoto City, Japan. 5. Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan. 6. Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark. 7. 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.
Abstract
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. CONCLUSION: 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. GENERAL SIGNIFICANCE: N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.
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 humanserum 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. CONCLUSION: 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. GENERAL SIGNIFICANCE: N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.
Authors: Tahia H Saleem; Nagwa Abo El-Maali; Mohammed H Hassan; Nahed A Mohamed; Nashwa A M Mostafa; Emaad Abdel-Kahaar; Azza S Tammam Journal: Int J Hepatol Date: 2018-09-02