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. 2022 Jun 10;10(6):1379. doi: 10.3390/biomedicines10061379

Figure 2.

Figure 2

Reaction specificity of wildtype and mutant recombinant mouse Alox15b. Wildtype and mutant (Tyr603Asp + His604Val) Alox15b were expressed as N-terminal his-tag fusion proteins as described in Section 2 and aliquots of the bacterial lysis supernatants were used as enzyme source. After a 10 min incubation period with exogenous AA (100 µM), the reaction products were analyzed with RP-HPLC, as reported in Section 2. (A) Representative partial RP-HPLC chromatogram of the AA oxygenation products formed by wildtype mouse Alox15b. Retention time of authentic standards are indicated by the arrows above the traces. Inset: UV-spectrum of the conjugated dienes eluting with a retention time of 8.5 min. (B) Representative partial RP-HPLC chromatogram of the AA oxygenation products formed by the Tyr603Asp + His604Val double mutant of mouse Alox15b. Inset: LC-MS analyses of the AA oxygenation products formed by wildtype and mutant Alox15b. (C) Statistical evaluation of the major AA oxygenation products formed by wildtype and mutant mouse Alox15b (n = 4).