Avidin biotin mixture

FIGURE 12 (A) ESI mass spectra of avidin (top) and of avidin - biotin mixture (bottom). Both the... 

Smith et al. studied the binding of biotin and some biotin derivatives to avidin and strepavidin.38 In the initial experiment, they measured the ESI mass spectrum of avidin (Fig. 12A) observing four charge states (15 + to 18 +) corresponding to a molecular mass of 63,915 Da which correlates well with the expected mass of 63,870 Da. The spectrum obtained for a mixture of avidin-biotin (lower plot) shows that peaks are shifted to higher m/z values that correspond to a mass increase of 973 u. This indicated that four biotin molecules are indeed bound to avidin. The mass increase is accompanied by a reduction of one unit in the charge states, which now range from 14+ to 17 +. This study demonstrated that the ESI process transfers the solution complex to the gas phase. 

To investigate the cofactor requirement and the characteristics of the enzyme, the effects of additives were examined using phenylmalonic acid as the representative substrate. The addition of ATP or ADP to the enzyme reaction mixtures, with or without coenzyme A, did not enhance the rate of reaction. From these results, it is concluded that these co-factors are not necessary for this decarboxylase. It is well estabhshed that avidin is a potent inhibitor of the bio-tin-enzyme complex [11 -14]. In the present case, addition of avidin has no influence on the decarboxylase activity, indicating that the AMDase is not a biotin enzyme. Thus, the co-factor requirements of AMDase are entirely different from those of known analogous enzymes, such as acyl-CoA carboxylases [15], methyhnalonyl-CoA decarboxylases [11] and transcarboxylases [15,16]. 

Mixture of D-penicillamine (pen, Fig. 22) and copper(II) chloride forms a complex of the formula [Cu(I)8Cu(II)6pen12]5-. The Ri value of this complex at 300 MHz and 23°C is 0.586 mM 1 s-1 [178]. The carboxylate groups on the cluster have been shown to react with aziridines such as XAMA-7 (Fig. 23) which in turn can be reacted with biotin [179]. The copper clusters can then be attached to antibodies or other biotinylated proteins via an avidin linker molecule. 

Fig. 12 Emission spectra of complex [Ir(ppy)2(dpq-biotin)]+ (22 pM) in a mixture of potassium phosphate buffer (50 mM, pH 7.4) and methanol (9 1, v/v) in the absence (broken line) and presence (solid line) of avidin (5.5 pM) [66]...

In the future, compounds may be recovered on scale by selective adsorption to resins (resin capture). In this way the product could be fished out from a mixture of compounds, even if the product were there in relatively dilute conditions. The biochemical analogy is affinity chromatography, e.g., the isolation of biotin and biotin-containing compounds by adsorption to avidin. In a recent example, propranolol (38) was fished out from a crude preparation by binding to... 

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