Conjugate formation

Awad, H. M. Boersma, M. G. Boeren, S. van Bladeren, P. J. Vervoort, J. Rietjens, I. M. C. M. Quenching of quercetin quinone/quinone methides by different thiolate scavengers stability and reversibility of conjugate formation. Chem. Res. Toxicol. 2003,16, 822-831. 

Figure 19.9 Conjugation of the biological peptide [Met5]-enkephalin to BSA using EDC. The graph shows the gel filtration profile (on Sephadex G-25) after completion of the conjugation reaction. A blank run with no added EDC was done to illustrate the peak absorbance that would be obtained if no conjugation took place. With addition of 10 mg of EDC to a reaction mixture consisting of 2 mg of BSA plus 2 mg of peptide, nearly complete conjugate formation was obtained.

Figure 19.10 To illustrate the consistency of an EDC-mediated reaction, [Met5]-enkephalin was conjugated to OVA using conditions identical to those described for BSA in Figure 19.9. Note the similarity in the degree of conjugate formation.

Figure 19.25 Absorbance scan comparing unconjugated cBSA with the same carrier that had been coupled with phenol red using the Mannich reaction. Two different reaction times are compared, indicating that extended reactions yield increased conjugate formation.

Antibody-enzyme conjugate formation through thioether bond... 

Dekant, W., 1993, Bioactivation of nephrotoxins and renal carcinogens by glutathione S-conjugate formation. Toxicol. Lett. 67 151-160 Dooley, D.M., 1999, Stmcture and biogenesis oftopaquiones and related cofactors. J. Biol. Inorg. Chem. 4 1-11... 

Glucuronide synthesis is the rate-determining step in hepatic bilirubin metabolism. Drugs such as phenobarbital, for example, can induce both conjugate formation and the transport process. 

Phase II reactions (conjugate formation). Type II reactions couple their substrates (bilirubin, steroid hormones, drugs, and products of phase I reactions) via ester or amide bonds to highly polar negatively charged molecules. The enzymes involved are transferases, and their products are known as conjugates. 

Studies of UDP-glucuronyltransferase with bilirubin as the acceptor substrate are technically difficult. This is indicated by frequent modification of the initial assay systems (A8, G9, L4, S4) and by the wide range of reported enzyme activities (Table 1). Possible causes of these discrepancies, which are manyfold, will be discussed in some detail below. The conclusions drawn should be helpful in the design of assays of conjugate formation of bilirubin and of the synthesis of mono- and diconjugates. 

With fresh rat liver homogenates that were not fortified with UDP-sugar, appreciable conjugate formation of bilirubin occurred (HIO, M5). The process occurred in the absence of added Mg-+ and was inhibited by digitonin (HIO). The conjugation rates at 37°C were constant for the first 3- to 5-minute period, then decreased gradually to zero (M5). 

To obtain blank values it is not suflScient to treat a duplicate incubation mixture with so-called diazo-blank reagent (NaN02 omitted from the diazo reagent), as is common practice in assaying serum or bile (H12). Contributions (b)-(d) would be obtained with the test, not with the control mixture. A control incubation mixture, treated with diazo reagent in parallel with the text mixture, will adequately measure contributions (b)-(f). Whether endogenous conjugate formation in the incubation control (contribution a) is suppressed completely or not, must depend on the preference of the reader (Sections 3.1.4 and 3.2). 

If (a) conjugate formation in the control incubation mixture is unimportant or can be suppressed (Section 3.2), (b) blank contributions are adequately determined, and (c) diazo-coupling of synthetic conjugates is reasonably complete, the difference in extinction of test and control (usually measured at 530-550 nm) allows the amount of pigment synthesized, and thus the value of BCR, to be obtained. 

C9. Cooke, B. A., and Taylor, W., The metabolism of progesterone by animal tissues in vitro. 4. Conjugate formation during the metabolism of (4- C) progesterone by female-rat liver homogenate. Biochem. J. 86, 365-371 (1963). 

---