N-acetyl-L-methionine is a superior protectant of human serum albumin against post-translational oxidation as compared to N-acetyl-L-tryptophan

Abstract

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.

Keywords: CD, circular dichroism; CT, chloramine-T; DSC, differential scanning calorimetry; HSA, human serum albumin; Human serum albumin; N-AcMet, N-acetyl-L-methionine; N-AcTrp, N-acetyl-L-tryptophan; N-Acetyl-L-Methionine; N-Acetyl-L-Tryptophan; Oct, octanoate; Oxidation; Pharmacokinetics; ROS, reactive oxygen species; Structural Changes; rHSA, recombinant HSA.

Graphical abstract

Fig. 1

AOPP content of non-treated rHSA (control) and oxidized rHSAs. The concentration of rHSA was 100 µM. Each column represents the mean ± S.D. (n=4). ^⁎⁎P<0.01, compared with control. ^##P<0.01, compared with CT-rHSA additive (-). ^††P<0.01, compared with CT-rHSA + N-AcTrp.

Fig. 2

(A) Carbonyl content of non-treated rHSA (control) and oxidized rHSAs. The concentration of rHSA was 0.2 mM and that of each additive was 1 mM. Each column represents the mean ± S.D. (n =3). ^⁎⁎P<0.01, compared with control. ^##P<0.01, compared with CT-rHSA additive (-). ^††P<0.01, compared with CT-rHSA + N-AcTrp. (B) Native-PAGE electrophoresis. The concentration of rHSA was 0.1 mM and that of each additive was 0.5 mM. (C) Western blots and staining with Oxyblot Kit reagents. (D) Level of rHSA oxidation as determined by densitometry ratios between DNP area and corresponding protein area. Each column represents the mean ± S.D. (n=4). ^##P<0.01, compared with CT-rHSA additive (-). ^††P<0.01, compared with CT-rHSA + N-AcTrp.

Fig. 3

Relative peroxynitrite scavenging activity of non-treated rHSA (control) and oxidized rHSAs. The concentration of rHSA was 25 µM. Each column represents the mean ± S. D. (n=3). ^⁎⁎P<0.01, compared with control. ^##P<0.01, compared with CT-rHSA additive (-). ^††P<0.01, compared with CT-rHSA + N-AcTrp.

Fig. 4

Relative hydroxyl radical scavenging activity of non-treated rHSA (control) and oxidized rHSAs. The concentration of rHSA was 50 µM. Each column represents the mean ± S. D. (n=3). ^⁎⁎P<0.01, compared with control. ^##P<0.01, compared with CT-rHSA additive (-). ^††P<0.01, compared with CT-rHSA + N-AcTrp.

Fig. 5

Thermogram of non-treated rHSA (control) and oxidized rHSAs obtained by DSC. (1) control, (2) CT-rHSA additive (-), (3) CT-rHSA + N-AcMet, (4) CT-rHSA + N-AcTrp dissolved in sodium phosphate buffer, pH 7.4. The protein concentration was 20 µM.

Fig. 6

Far-UV CD spectra of non-treated rHSA (control) and oxidized rHSAs. (1) control, (2) CT-rHSA additive (-), (3) CT-rHSA + N-AcMet, (4) CT-rHSA + N-AcTrp dissolved in sodium phosphate buffer, pH 7.4. The protein concentration was 10 µM.

Fig. 7

Intrinsic fluorescence spectra of non-treated rHSA (control) and oxidized rHSAs. (1) control, (2) CT-rHSA additive (-), (3) CT-rHSA + N-AcMet, (4) CT-rHSA + N-AcTrp dissolved in sodium phosphate buffer, pH 7.4. The protein concentration was 2 µM. au, arbitrary units.

Fig. 8

In vivo pharmacokinetics of ICG-labeled, non-treated rHSA (control) and oxidized rHSAs. Each data point represents the mean ± S.D. (n=6). ^⁎⁎P<0.01, compared with control. ^##P<0.01, compared with CT-rHSA additive (-). ^††P<0.01, compared with CT-rHSA + N-AcTrp.