NMR and MS analysis of decomposition compounds produced from N-acetyl-L-glutamine at low pH.

N-Acetyl-L-glutamine decomposition products glutamine, glutamic acid, pyroglutamic acid, N-acetylglutamic acid, and a novel compound, N-(2,6-dioxo-3-piperidinyl) acetamide, have been identified by NMR and MS techniques. N-Acetylglutamine, a modified amino acid, offers greater chemical stability than glutamine in conditions that are experienced during typical sterilization and shelf storage of liquid nutritionals. However, to support safety and stability studies, potential decomposition products of N-acetyl-L-glutamine needed to be identified. Therefore, atypically harsh conditions were used; an unbuffered (pH <3) 1 mg/mL water solution of N-acetyl-L-glutamine was heated to 100 degrees C for 3 h. One-dimensional proton and proton-decoupled carbon-13 NMR and electrospray LC-MS/MS techniques were employed to identify the molecular structures of the generated N-acetyl-L-glutamine decomposition products. Additionally, DEPT and two-dimensional NMR techniques TOCSY, GMQCOSY, GHSQC, and GHMBC were employed to derive the final structure of the acetamide.

Effects of Zn(II) on galactosyltransferase activity.

The enzyme beta-4-galactosyltransferase (GT) catalyzes the transfer of a galactosyl group from UDP-galactose to N-acetylglucosamine (GlcNAc) on glycoproteins. In the presence of alpha-lactalbumin (alpha-LA), galactosyltransferase catalyzes the transfer of galactose to glucose to yield lactose. It is known that, in the absence of alpha-lactalbumin, Zn(II) competes with Mn(II) for the same binding site(s) in galactosyltransferase, resulting in an increase in the apparent Michaelis constant, Km(app), for Mn(II)-activation of N-acetyllactosamine synthesis. In the presence of alpha-lactalbumin (i.e., lactose synthase), the Mn(II)-activation is biphasic and the initial phase is inhibited by increasing concentrations of Zn(II). The Zn(II) inhibition of lactose synthase plateaus at [Zn(II)]:[alpha-lactalbumin] approximately 1:1, while for N-acetyllactosamine synthesis there is no plateau at all. The results suggest that Zn(II) binding to alpha-lactalbumin effects lactose synthase. Kinetically, Zn(II) induces a decrease in both the Km(app) and Vm for Mn(II), which results in an apparent increase, followed by a decrease, in lactose synthase activity at Mn(II) concentrations below saturation of the first [Mn(II)] binding site. Increasing Zn(II) also decreases Km(app) and Vm for both glucose and UDP-galactose in the lactose synthase reaction with either both Ca(II)- or apo-alpha-lactalbumin, further suggesting novel interactions between Zn(II)-alpha-lactalbumin and the lactose synthase complex, presumably mediated via a Zn(II)-induced conformational change upon binding to alpha-lactalbumin. On the other hand, in N-acetyllactosamine synthesis, Zn(II) only slightly effects Km(app) for N-acetylglucosamine and has essentially no effect on Km(app) or Vm for UDP-galactose.

Binding of Zn(II) ions to alpha-lactalbumin.

The binding of Zn(II) ions to human and bovine alpha-lactalbumin has been studied by fluorescence, scanning microcalorimetry, and proteolytic digestion. The intrinsic tryptophan fluorescence spectrum of Ca(II)-loaded alpha-lactalbumin is insensitive to Zn(II) binding to the strong cation binding sites (Zn:protein ratios up to 20), yet the thermal denaturation transition, as detected by intrinsic fluorescence, is shifted toward lower temperatures. On the other hand, low concentrations of Zn(II) ([Zn]:[protein] less than 1) shift heat sorption curves toward lower temperatures. It was concluded that alpha-lactalbumin possess several relatively strong Zn(II) binding sites, which are filled sequentially, the process being accompanied by protein aggregation. The strongest Zn(II) binding (5 x 10(5) M-1) increases its susceptibility to tryptic and chymotryptic digestion, slightly decreases its affinity for the fluorescent probe, bis-ANS, and alters its interactions with UDP-galactose. Zn(II) binding to aggregated forms of alpha-lactalbumin increases its affinity to bis-ANS.