Oxidation carbohydrate amines

Note At a level of 50-pl probe addition, polyclonal human IgG will be modified at a level that gives an F/P ratio of about 0.113. Since the labeling occurs only at the oxidized carbohydrate sites, the fluorophore incorporation typically is less than that observed when using amine-reactive probes. 

The ethylene diamine-dextran derivative may be used for the coupling of carboxylate-contain-ing molecules by the carbodiimide reaction, for the coupling of amine-reactive probes, or to modify further using heterobifunctional crosslinkers. The hydrazide-dextran derivative may be used to crosslink aldehyde-containing molecules, such as oxidized carbohydrates or glycoproteins. 

Another function of ascorbic add is to generate Fe(II) from Fe (III), which is part of the Haber-Weiss-Fenton Reaction (77). In that reaction, peroxide is reduced to the hydroxyl radical, HO, an extremely powerful oxidizing agent. It should also be noted that ascorbic acid can chelate metals 18 and will promote the carbohydrate-amine browning reaction (79-20). 

Dennis Johnson and co-workers pioneered the area of pulsed electrochemical detection (PAD) [30,31]- In this technique, electrode fouling is circumvented through the use of a triple-pulse waveform. Alcohols, carbohydrates, amines, and sulfur compounds can all be detected using this technique. Alcohols and carbohydrates are determined by direct oxidation at the regenerated electrode surface, while amines and sulfur compounds undergo an oxide-catalyzed oxidation. 

The pulsed amperometric detector (PAD) developed by Johnson and co-workers using an Au or Pt electrode has permitted the direct detection of aliphatic alcohols including carbohydrates, amines, and sulfur compounds. Fouling of these electrodes is prevented by application of both positive (to eliminate sample adsorption) and negative (to reduce any metal oxide) reactivation step potentials on the order of 100 ms before resetting the potential for detection of the analyte. The analytical current is usually sampled near the end of the detection potential pulse to permit decay of the charging current. The oxidation of these aliphatic compounds such as carbohydrates is facilitated in basic solution at about pH 12, so postcolumn addition of 0.1 Af NaOH or the use of a polymeric column with a basic mobile phase is required. Detection limits of alcohols and carbohydrates are at the 10 ppb level. Alka-nolamines, amino acids, and sulfur compounds other than sulfonic acids and sulfones can also be detected. 

Apart from the amino group modification, various strategies for protein modification have been published. PEG-amine (Fig. 3j) is also utilized for the modification of bioactive compounds with carboxyl groups [38,39] and for a highly reactive intermediate of various modifier syntheses [40] such as PEG-maleimide (Fig, 3k). Urrutigoity et al. [41] coupled PEG-amine to the periodate-oxidized carbohydrate moiety of a glycoprotein. 

Hepatic steatosis usually is a result of excessive administration of carbohydrates and/or lipids, but deficiencies of carnitine, choline, and essential fatty acids also may contribute. Hepatic steatosis can be minimized or reversed by avoiding overfeeding, especially from dextrose and lipids.35,38 Carnitine is an important amine that transports long-chain triglycerides into the mitochondria for oxidation, but carnitine deficiency in adults is extremely rare and is mostly a problem in premature infants and patients receiving chronic dialysis. Choline is an essential amine required for synthesis of cell membrane components such as phospholipids. Although a true choline deficiency is rare, preliminary studies of choline supplementation to adult patients PN caused reversal of steatosis. 

This simple strategy can be used to add amine residues to polysaccharide molecules after formation of aldehydes by periodate or enzymatic oxidation (Section 4.4, this chapter). Thus, glycoconjugates or carbohydrate polymers such as dextran may be derivatized to contain amines for further conjugation reactions. 

Figure 20.14 Periodate oxidation of HRP creates aldehyde groups on the carbohydrate chains of the enzyme. Reaction with a Fab fragment then may be done using reductive amination to produce a lower-molecular-weight complex than would be obtained using intact IgG antibodies.

Conjugation of HRP by reductive amination can be done by oxidizing the carbohydrate on the enzyme and subsequently coupling to the amines on (strept)avidin (Figure 23.5). 

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