Figure 3. Cδ¹ of tryptophan is the site of methylene bridge acylation.

(A) Proposed mechanisms for methylene bridge-tryptophan adduct formation. After induction by other free radicals, methylene bridge free radicals may attack either Cδ¹ and Nɛ¹ of tryptophan to form either adduct I or II, respectively.

(B) Free radical inducer promotes tryptophan acylation. The formation of EPA, DHA, and LA adducts in a peptide with or without ammonia persulfate in thereaction mix was analyzed by MS (upper), and adducts levels were measured (lower) (n = 3, mean ± SEM).

(C) Free radical scavenger inhibits tryptophan acylation. The formation of the EPA, DHA, and LA adduct in a peptide with or without ascorbic acid in the reaction mix was analyzed by MS (upper), and adducts levels were measured (lower) (n = 3, mean ± SEM).

(D) Cδ¹ of tryptophan is the acylation site by a methylene bridge. A tryptophan-containing peptide (Ac-FTEGAFKDWGYQLA) and peptides with the same sequence but with tryptophan replaced by Nɛ¹ or Cδ¹ methyl-blocked tryptophan were tested for their abilities to be adducted by linoleic acid, EPA, and DHA. Adduct formation was assayed by MS.

(E) NMR analysis of the reactivities of Cδ¹ of tryptophan. The NMR signal of Cδ¹ of tryptophan was decreased by LA (left), DHA (middle), and EPA (right).

(F and G) AKT signaling activation by DHA was reversed by tryptamine (F) and 5-hydroxy-tryptamine (5-HT) (G). The levels of p-AKT2(T308) and p-AS160(T642) were determined in 3T3-L1 cells cultured in media supplemented with DHA, DHA/tryptamine, or DHA/5-HT.