Stabilizing mechanisms in commercial albumin preparations: octanoate and N-acetyl-l-tryptophanate protect human serum albumin against heat and oxidative stress |
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Authors: | Makoto Anraku Yasufumi Tsurusaki Hiroshi Watanabe Toru Maruyama Ulrich Kragh-Hansen Masaki Otagiri |
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Affiliation: | 1. Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan;2. Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark |
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Abstract: | The capability of octanoate, N-acetyl-l-tryptophanate (N-AcTrp) and other ions of fatty acids and amino acids to stabilize human serum albumin (HSA) against thermal and oxidative stress was studied. Native-PAGE showed that octanoate, and more hydrophobic fatty acids anions, stabilizes the monomeric form of HSA during heating at 60 °C for 30 min. Heating in the presence of octanoate did not change the far-UV CD-spectrum. The stabilizing role of octanoate also showed as an increase in denaturation temperature and calorimetric enthalpy, determined by differential scanning calorimetry (DSC). N-AcTrp, which was found to compete with octanoate for a common high-affinity site, has only a minor stabilizing effect. By contrast, no effect was found for l-tryptophanate or N-acetyl-l-cysteinate. Any ligand effect on oxidation was examined by using 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH) as oxidizing agent. One hour of incubation resulted in the formation of the same number of carbonyl groups, whether octanoate or one of the abovementioned amino acids was present or not. However, the number of groups formed after 24 h of incubation was significantly decreased in the presence of l-tryptophanate and, especially, N-AcTrp. The effect of 1-h incubation with AAPH on the oxidative status of 34-Cys was studied by the HPLC technique. It was found that N-AcTrp, but not octanoate, has a large protecting effect on the sulfhydryl group. Thus, octanoate has the greatest stabilizing effect against heat, whereas the presence of N-AcTrp diminishes oxidation of HSA. |
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Keywords: | HSA human serum albumin HMA mercaptalbumin HNA-1 albumin forming mixed disulfides HNA-2 sulfhydryl-oxidized albumin Oct octanoate AAPH 2,2′-azobis(2-amidino-propane)dihydrochloride DSC differential scanning calorimetry CD circular dichroism Human serum albumin Sulfhydryl groups Thermal stability Antioxidant activity Octanoate Corresponding author. Tel.: +81 96 371 4150 fax: +81 96 362 7690. |
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