RESUMEN
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.
Asunto(s)
Depuradores de Radicales Libres/química , Metionina/análogos & derivados , Especies Reactivas de Oxígeno/química , Albúmina Sérica/química , Triptófano/análogos & derivados , Humanos , Metionina/química , Oxidación-Reducción , Procesos Fotoquímicos , Estabilidad Proteica , Triptófano/químicaRESUMEN
Sodium octanoate and N-acetyl-L-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, as compared with N-AcTrp, N-acetyl-L-methionine (N-AcMet) is superior in protecting albumin exposed to light during storage. Here, we examine, whether N-AcMet also is better than N-AcTrp to protect albumin against oxidation. Recombinant human serum albumin (rHSA) without and with N-AcMet or N-AcTrp was oxidized by using chloramine-T (CT) as a model compound for mimicking oxidative stress. Oxidation of rHSA was examined by determining carbonyl groups and advanced oxidation protein products. Structural changes were studied by native-PAGE, circular dichroism, intrinsic fluorescence and differential scanning calorimetry. The anti-oxidant capacity of CT-treated rHSA was quantified by its ability to scavenge peroxynitrite and the hydroxyl radical. The pharmacokinetics of indocyanine green-labeled albumin preparations was studied in male mice. We found that the number of chemical modifications and the structural changes of rHSA were significantly smaller in the presence of N-AcMet than in the presence of N-AcTrp. The anti-oxidant properties of CT-exposed rHSA were best protected by adding N-AcMet. Finally, N-AcMet is superior in preserving the normal pharmacokinetics of rHSA. Thus, N-AcMet is superior to N-AcTrp in protecting albumin preparations against oxidation. In addition, N-AcMet is probable also useful for protecting other proteins. Therefore, N-AcMet should be useful as a new and effective stabilizer and antioxidant for albumin isolated from blood, rHSA, albumin-fusion proteins and for preparations of rHSA-therapeutic complexes.