Avidin

As a result of impaired activity of acetyl CoA and propionyl CoA carboxylases, there are changes in the fatty acid composition of lipids in the lymphocytes of biotin-deficient rats. There is an increase in the proportion of long-chain fatty acids (C22 0 to C30 0) and odd-carbon fatty acids (Cl 5 0 to C29 0), with a decrease in the proportion of unsaturated fatty acids and the ratio of ds-vaccenic acid (C18 l )9) palmitoleic acid (C16 lft)6), which is indicative of impaired elongation and desaturation of fatty acids (Liu et al., 1994). 

It is apparent from the discussion in Section 11.3 that there is Utde information concerning human biotin requirements and no evidence on which to base recommendations. Average intakes of biotin range between 15 to 70 /rg per day. Such intakes are obviously adequate to prevent deficiency, and the safe and adequate range of biotin intakes is set at 10 to 200 /xg per day (Department of Health, 1991 Scientific Committee for Food, 1993). The U.S./Canadian adequate intake for adults is 30 /xgper day (Institute of Medicine, 1998). 

On the basis of studies in patients who developed deficiency during total parenteral nutrition, and who are therefore presumably wholly reliant on an exogenous source of the vitamin - with no significant contribution from intestinal bacterial synthesis - the provision of 60 fxg of biotin per day for adults receiving total parenteral nutrition is generally recommended (Bitsch et al., 1985). 

The original interest in avidin was because of the egg white injury that was subsequently shown to be avidin-induced biotin deficiency. Thereafter, avidin was used because of its high affinity for biotin (a dissociation constant of 10 mol per L), not only to induce experimental biotin deficiency, but also to bind to biotin in isolated enzymes and thus, by irreversible inhibition, demonstrate the coenzyme role of biotin. Because of the stability of the avidin-biotin complex, it has not been possible to use immobilized avidin as a means of purifying biotin enzymes - there seems to be no way in which the enzyme can be released from avidin binding. Because of its high affinity for biotin, avidin is used to provide an extremely sensitive system for linking reporter molecules in a variety of analytical systems. 

Avidin has been found in the eggs and oviducts of many species of birds and in the egg jelly of frogs, but not in other tissues and not in the mammalian 

In conclusion, both mono-PEGylated interferons present superior properties for protein delivery, resulting in a longer-lasting activity as compared to the native protein, avoiding frequent administration, and improving the therapeutic performance of interferon. 

It has been suggested that the modification of the protein s isoelectric point could result in an alteration of its pharmacokinetic profile. Avidin acylation was performed by lysine amino group derivatization with succinyl anhydride or other anhydrides, which allowed the isoelectric point to be shifted to more acidic values, depending on the level of modification. Indeed, the protein anionization induced a reduction of accumulation in the liver, but resulted only in a limited prolongation of residence time in the circulation [30, 31]. 

In a recent study, avidin was extensively modified with linear 5- and 10-kDa PEGs and with a branched 20-kDa PEG. In order to maintain high biological activity, the polymer was conjugated in the presence of a macromolecular active site protective agent, which was used to avoid polymer attachment on the protein area 

Thermoresponsive acrylamide co-polymers were also used to alter the physicochemical and biopharmaceutical properties of avidin. Similar to PEG, the acrylamide co-polymers with a lower critical solution temperature (LCST) of about 37 °C were conjugated to the protein amino groups. The polymers were conjugated either by polymer multipoint attachment using polyfunctional polymers or by single chain attachment using end-chain monoactivated polymer. In both cases, the polymer conjugation was found to produce bioactive derivatives with reversible thermal character (Fig. 11.12). 

A closely similar protein, streptavidin, has been isolated from culture filtrates of several species of Streptomyces. Unlike avidin, streptavidin is not glycosylated and has an acidic isoelectric point. It binds biotin with a similarly high affinity. 

5 Ovoglobulins G2 and G3 These proteins are good foam builders. 

This protein, of which three components are known, can apparently form fibrillar structures and so contribute to a rise in viscosity of albumen, particularly of the thick, gel-like egg white (see egg structure. Fig. 11.1), where it occurs in a four-fold higher concentration than in fractions of thin albumen. 

Ovomucin has been separated into a low-carbohydrate (carbohydrate content ca. 15%) a-fraction and a high-carbohydrate (carbohydrate content ca. 50%) -fraction. It appears to be associated with polysaccharides. The compositions of its carbohydrate moieties are given in Table 11.5. Ovomucin is heat stable. It forms a water-insoluble complex with lysozyme. The dissociation of the complex is pH dependent. Presumably it is of importance in connection with the thinning of egg white during storage of eggs. 

Avidin is a basic glycoprotein (Table 11.5). Its amino acid sequence has been determined. Noteworthy is the finding that 15 positions (12% of the total sequence. Table 11.7) are identical with those of lysozyme. Avidin is a tetramer consisting of four identical subunits, each of which binds one mole of biotin. The dissociation constant of the avidin-biotin complex at pH 5.0 is k i/ki = 1.3 x 10 mol/1, i. e., it is extremely low. The free energy and free enthalpy of complex formation are AG = — 85kJ/mole and AH = —90kJ/mole, respectively. Avidin, in its form in egg white, is practically free of biotin, and presumably fulfills an antibacterial role. Of interest is the occurrence of a related biotin-binding protein (streptavidin) in Streptomyces spp., which has antibiotic properties. 

---

Fig. XV-5. Fluorescence micrographs illustrating morphologies of two-dimensional (2D) sireptavidin crystals at three streptavidin/avidin ratios 15/85, 25/75, 40/60 from left to ri t. Scale bar is 100 gm (From Ref. 31.)...

A typical force curve showing the specific avidin-biotin interaction is depicted in figure Bl.20.10. The SFA revealed the strong influence of hydration forces and membrane undulation forces on the specific binding of proteins to membrane-bound receptors [81]. 

The avidin-biotin complex, known for its extremely high affinity (Green, 1975), has been studied experimentally more extensively than most other protein-ligand systems. The adhesion forces between avidin and biotin have been measured directly by AFM experiments (Florin et al., 1994 Moy et al., 1994b Moy et al., 1994a). SMD simulations were performed on the entire tetramer of avidin with four biotins bound to investigate the microscopic detail of nnbinding of biotin from avidin (Izrailev et al., 1997). 

The simulations also revealed that flapping motions of one of the loops of the avidin monomer play a crucial role in the mechanism of the unbinding of biotin. The fluctuation time for this loop as well as the relaxation time for many of the processes in proteins can be on the order of microseconds and longer (Eaton et al., 1997). The loop has enough time to fluctuate into an open state on experimental time scales (1 ms), but the fluctuation time is too long for this event to take place on the nanosecond time scale of simulations. To facilitate the exit of biotin from its binding pocket, the conformation of this loop was altered (Izrailev et al., 1997) using the interactive molecular dynamics features of MDScope (Nelson et al., 1995 Nelson et al., 1996 Humphrey et al., 1996). 

The simulations of the avidin-biotin complex (Izrailev et ah, 1997) showed that a major difficulty involved in studies of the binding and flexibility of... 

Izrailev et al., 1997] Izrailev, S., Stepaniants, S., Balsera, M., Oono, Y., and Schulten, K. Molecular dynamics study of unbinding of the avidin-biotin complex. Biophys. J. 72 (1997) 1568-1581... 

Thus, we have found unexpected complexities and even in this simple system have not yet been unable to accurately extrapolate the results of simulations done over periods varying from 1 to several hundred ps, to the low-friction conditions of extraction experiments performed in times on the oi dc r of ms. The present results indicate that one should not expect agreement between extraction experiments and simulations in more complex situations typically found in experiments, involving also a reverse flow of water molecules to fill the site being evacuated by the ligand, unless the simulation times are prolonged well beyond the scope of current computational resources, and thereby strengthen the conclusion reached in the second theoretical study of extraction of biotin from it.s complex with avidin [19]. 

Proteias, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc iaclude bovine and human semm albumin, a -acid glycoproteia, ovomucoid, avidin, and ceUobiohydrolase. The bovine semm albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human semm albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a -acid glycoproteia and ceUobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

Isotope Dilution Assay. An isotope dilution assay for biotin, based on the high affinity of avidin for the ureido group of biotin, compares the binding of radioactive biotin and nonradio active biotin with avidin. This method is sensitive to a level of 1—10 ng biotin (82—84), and the radiotracers typically used are p C]biotin (83), [3H]biotin (84,85) or an I-labeled biotin derivative (86). A variation of this approach uses I-labeled avidin (87) for the assay. 

Avidin (from egg white) [1405-69-2] Mr -70,000. Purified by chromatography of an ammonium acetate soln on CM-cellulose [Green Biochem J 101 774 1966]. Also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr 7 Bio/C/iew 256 761 1981]. It is a biotin binding protein. 

Avidin-biotinic system as the aid of address delivery of antitumor compounds 98MI54. 

Alimentary biotin deficiency is rare. It may, however, occur in patients on long-term parenteral nutrition lacking biotin or in persons who frequently consume raw egg white. Raw egg white contains a biotin-binding glycoprotein, called avidin, which renders biotin biologically unavailable. Pharmacological doses of the vitamin (1-10 mg/d) are then used to treat deficiency symptoms. There are no reports of toxicity for daily oral doses up to 200 mg and daily intravenous doses of up to 20 mg [2]. 

These examples are part of a broader design scheme to combine catalytic metal complexes with a protein as chiral scaffold to obtain a hybrid catalyst combining the catalytic potential of the metal complex with the enantioselectivity and evolvability of the protein host [11]. One of the first examples of such systems combined a biotinylated rhodium complex with avidin to obtain an enantioselective hydrogenation catalyst [28]. Most significantly, it has been shovm that mutation-based improvements of enantioselectivity are possible in these hybrid catalysts as for enzymes (Figure 3.7) [29]. 

Metagenomics has been applied to the search for novel genes encoding the synthesis of vitamins such as biotin and vitamin C [47,76]. Seven cosmids were detected in metagenomic libraries obtained after avidin enrichment of environmental samples. The highest levels of biotin production in this study were detected in a cosmid... 

---