Biotin reagents for antibody pretargeting. 7. Investigation of chemically inert biotinidase blocking functionalities for synthetic utility

An investigation was conducted to evaluate three biotin derivatives designed to block biotinidase cleavage of the biotinamide bond. Difficulties in multistep syntheses of molecules containing tert-butyl protected hydroxymethyl and carboxylate groups positioned alpha to a biotinamide bond led to the investigation of alternative biotinidase-blocking moieties that do not require protection and deprotection. The targeted biotin derivatives contained serine-O-methyl ether, 2-aminobutyric acid, and valine moieties conjugated to the biotin carboxylate functionality. Those derivatives were further modified with a radioiodinated aryl ring to study their biotinidase stability. As a comparison to previously studied biotin derivatives, radioiodinated versions of biotin conjugates that contained (a) no biotinidase stabilizing group, (b) an N-methyl (sarcosine) stabilizing group, (c) an alpha-carboxylate (aspartate) stabilizing group and hydroxymethyl (serine) stabilizing group were also prepared and tested. When tested in human serum, all of the radioiodinated biotinidase-stabilized biotin derivatives had <1% biotinamide cleavage. Thus, under the conditions studied, all of the tested biotinidase blocking moieties appeared to be equal with regards to protection from biotinidase cleavage. Further testing of the biotin derivatives included a HPLC assay to determine their relative dissociation from recombinant streptavidin (rSAv). The dissociation of cyanocobalamin (CN-Cbl) adducts of biotin-serine-O-methyl ether, biotin-aminobutyric acid, and biotin-valine were compared with the CN-Cbl adduct of biotin-sarcosine. The relative rates of dissociation found were biotin-sarcosine-CN-Cbl > biotin-valine-CN-Cbl > biotin-serine-O-methyl ether-CN-Cbl > biotin-aminobutyric acid-CN-Cbl. Due to the high cost of serine-O-ethyl ether (and its N-Boc derivative) and difficulty in syntheses of its biotin derivatives, that adduct is not an attractive candidate for application to compounds used in vivo. The higher lipophilicity and diminished binding of the biotin-valine adduct also makes its use in vivo less attractive. Thus, the biotin-aminobutyric acid adduct appears to be the best candidate for incorporation into biotin derivatives used in vivo, as it simplifies the synthetic procedures, has low cost, and provides effective blocking of biotinidase while retaining high binding affinity.     

Biotin reagents for antibody pretargeting. 4. Selection of biotin conjugates for in vivo application based on their dissociation rate from avidin and streptavidin

An investigation was conducted to determine the affect of structural variation of biotin conjugates on their dissociation rates from Av and SAv. This information was sought to help identify optimal biotin derivatives for in vivo applications. Fifteen biotin derivatives were conjugated with a cyanocobalamin (CN-Cbl) derivative for evaluation of their "relative" dissociation rates by size exclusion HPLC analysis. Two biotin-CN-Cbl conjugates, one containing unaltered biotin and the other containing iminobiotin, were prepared as reference compounds for comparison purposes. The first structural variations studied involved modification of the biotinamide bond with a N-methyl moiety (i.e., sarcosine conjugate), lengthening the valeric acid side chain by a methylene unit (i.e., homobiotin), and replacing the biotinamide bond with thiourea bonds in two conjugates. The rate of dissociation of the biotin-CN-Cbl derivative from Av and SAv was significantly increased for biotin derivatives containing those structural features. Nine additional biotin conjugates were obtained by coupling amino acids or functional group protected amino acids to the biotin moiety. In the conjugates, the biotin moiety and biotinamide bond were not altered, but substituents of various sizes were introduced alpha to the biotinamide bond. The results obtained from HPLC analyses indicated that the rate of dissociation from Av or SAv was not affected by small substituents alpha to the biotinamide (e.g., methyl, hydroxymethyl, and carboxylate groups), but was significantly increased when larger functional groups were present. On the basis of the results obtained, it appears that biotin conjugates which retain an unmodified biotin moiety and have a linker molecule conjugated to it that has a small functional group (e.g., hydroxymethylene or carboxylate) alpha to the biotinamide bond are excellent candidates for in vivo applications. These structural features are obtained in the biotin amino acid conjugates: biotin-serine, biotin-aspartate, biotin-lysine, and biotin-cysteine. Importantly, these biotin derivatives can be readily conjugated with other molecules for specific in vivo applications. In our studies, these derivatives will be used in the design of new biotin conjugates to carry radionuclides for cancer therapy using the pretargeting approach.     

A biotin-protein bond with stability in plasma

A nonradioactive label for peptide hormones would be useful for pharmacokinetic studies in infants, children, and pregnant women. Because the binding affinity between biotin and avidin is large (Ka=10(15) M(-1)), biotin could also serve as a covalent label for subsequent detection using a variety of avidin conjugates. However, biotin labels produced by most commercially available biotinylating reagents are rapidly cleaved from protein in plasma. We sought to synthesize a stable biotin label for protein. With the use of immunoglobulin G (IgG) as a model protein, biotin was conjugated through a cysteine residue; a carboxylate group was positioned alpha to the biotinamide bond. Stability of the bond in the presence of plasma and buffer control was assessed by release of biotin. Released biotin was separated from biotinylated IgG by ultrafiltration and was quantitated by an avidin-binding assay. In plasma, less than 0.6% of bound biotin was released. This release rate is not significantly different from buffer and is less than 7% of the release rate for IgG biotinylated by N-hydroxysuccinimide-LC-biotin. We conclude that this biotin-protein bond is stable in plasma. We speculate that many uses of avidin-biotin technology could be improved by using this method for protein labeling.     

Extension of the single amino acid chelate concept (SAAC) to bifunctional biotin analogues for complexation of the M(CO)3(+1) Core (M = Tc and Re): syntheses, characterization, biotinidase stability, and avidin binding

Biotin and avidin form one of the most stable complexes known (K(D) = 10(-15) M(-1)) making this pairing attractive for a variety of biomedical applications including targeted radiotherapy. In this application, one of the pair is attached to a targeting molecule, while the other is subsequently used to deliver a radionuclide for imaging and/or therapeutic applications. Recently, we reported a new single amino acid chelate (SAAC) capable of forming stable complexes with Tc(CO)3 or Re(CO)3 cores. We describe here the application of SAAC analogues for the development of a series of novel radiolabeled biotin derivatives capable of forming robust complexes with both Tc and Re. Compounds were prepared through varying modification of the free carboxylic acid group of biotin. Each 99mTc complex of SAAC-biotin was studied for their ability to bind avidin, susceptibility to biotinidase, and specificity for avidin in an in vivo avidin-containing tumor model. The radiochemical stability of the 99mTc(CO)3 complexes was also investigated by challenging each 99mTc-complex with large molar excesses of cysteine and histidine at elevated temperature. All compounds were radiochemically stable for greater than 24 h at elevated temperature in the presence of histidine and cysteine. Both [99mTc(CO)3(L6)]+1 [TcL6; L6 = biotinylamidopropyl-N,N-(dipicolyl)amine] and [99mTc(CO)3(L12a)]+1 (TcL12; L12 = N,N-(dipicolyl)biotinamido-Boc-lysine; TcL12a; L12a = N,N-(dipicolyl)biotinamide-lysine) readily bound to avidin whereas [99mTc(CO)3(L9)]+1 [TcL9; L9 = N,N-(dipicolyl)biotinamine] demonstrated minimal specific binding. TcL6 and TcL9 were resistant to biotinidase cleavage, while TcL12a, which contains a lysine linkage, was rapidly cleaved. The highest uptake in an in vivo avidin tumor model was exhibited by TcL6, followed by TcL9 and TcL12a, respectively. This is likely the result of both intact binding to avidin and resistance to circulating biotinidase. Ligand L6 is the first SAAC analogue of biotin to demonstrate potential as a radiolabeled targeting vector of biotin capable of forming robust radiochemical complexes with both 99mTc and rhenium radionuclides. Computational simulations were performed to assess biotin-derivative accommodation within the binding site of the avidin. These calculations predict that deformation of the surface domain of the binding pocket can occur to accommodate the transition metal-biotin derivatives with negligible changes to the inner-beta-barrel, the region most responsible for binding and retaining biotin and its derivatives. The biological activity and biodistribution of the technetium complexes TcL6, TcL9, and TcL12a were examined in an avidin tumor model. In the avidin bead tumor localization model, TcL6 demonstrated the most favorable localization with a 7:1 ratio of avidin bead implanted muscle versus normal muscle, while TcL9 exhibited a 2:1 ratio. However, TcL9 displayed no specificity for avidin.     

Neoglycoproteins: preparation and properties of complexes of biotinylated asparagine-oligosaccharides with avidin and streptavidin

Neoglycoproteins in which the oligosaccharide moieties are attached noncovalently to the protein through a high-affinity ligand have been prepared from biotinylated oligosaccharides and avidin or the nonglycosylated microbial analogue streptavidin. One of the asparagine-oligosaccharides purified from Pronase-digested ovalbumin (Man6-GlcNAc2-Asn) was reacted with an excess of the hydroxysuccinimide ester of biotin or, for the purpose of quantitation, [3H]biotin. Derivatives were also prepared with an extension "arm", a 6-aminohexanoyl group, between biotin and asparagine. When the purified biotinyl-Asn-oligosaccharide was added to avidin or streptavidin, a complex was formed containing 3 mol of oligosaccharide/mol of protein. The complexes were stable at neutral pH in the absence of biotin and could be dialyzed for 2 weeks without any significant loss of ligand. In the presence of biotin, or under denaturing conditions, the oligosaccharide derivative was released and could be quantitatively recovered. To assess the influence of the protein matrix on the reactivity of the oligosaccharide units, free biotinyl-Asn-oligosaccharide and the corresponding avidin and streptavidin complexes were exposed to alpha-mannosidase in parallel experiments. The rate of hydrolysis of the free derivative was severalfold faster than that of the two protein complexes, and at the time when about 90% of the free derivative had all five alpha-mannosyl residues removed, the majority of the protein-bound derivatives contained two to four undigested alpha-mannosyl residues and also had a significant amount of undigested starting material. The ease of preparation and the properties of these neoglycoproteins suggest that they should be excellent models for the study of glycoprotein-receptor binding and glycoprotein processing.     

Fourier-transform infrared spectroscopic studies on avidin secondary structure and complexation with biotin and biotin-lipid assemblies.

Fourier-transform infrared studies have been carried out to investigate the secondary structure and thermal stability of hen egg white avidin and its complexes with biotin and with a biotinylated lipid derivative, N-biotinyl dimyristoyl phosphatidylethanolamine (DMBPE) in aqueous dispersion. Analysis of the amide I stretching band of avidin yielded a secondary structural content composed of approximately 66% beta-sheet and extended structures, with the remainder being attributed to disordered structure and beta-turns. Binding of biotin or specific association with the biotinylated lipid DMBPE did not result in any appreciable changes in the secondary structure content of the protein, but a change in hydrogen bond stability of the beta-sheet or extended chain regions was indicated. The latter effect was enhanced by surface interactions in the case of the biotin-lipid assemblies, as was demonstrated by electrostatic binding to a nonspecific negatively charged lipid. Difference spectra of the bound biotin implicated a direct involvement of the ureido moiety in the ligand interaction that was consistent with hydrogen bonding to amino acid residues in the avidin protein. It was found that complexation with avidin leads to a decrease in bond length of the biotin ureido carbonyl group that is consistent with a reduction of sp3 character of the C-O bond when it is hydrogen bonded to the protein. Studies of the temperature dependence of the spectra revealed that for avidin alone the secondary structure was unaltered up to approximately 75 degrees C, above which the protein undergoes a highly cooperative transition to an unfolded state with concomitant loss of ordered secondary structure. The complexes of avidin with both biotin and membrane-bound DMBPE lipid assemblies display a large increase in thermal stability compared with the native protein.     

Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin

We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs based on lead compounds because it is difficult to predict the structural and conformational consequences on the resultant protein-ligand interactions.     

Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column

A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1h at 120 degrees C and the recovery for biocytin was 65.7+/-2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, "natto", rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.     

Urea amidolyase of Candida utilis. Characterization of the urea cleavage reactions.

Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources. The reactions involved in urea cleavage can be distinguished from one another on the basis of their sensitivities to (a) heat, (b) pH, and (c) chemical inhibitors. Evidence is presented for the product of the first reaction in urea cleavage, biotin carboxylation. Production of carboxylated enzyme is ATP dependent and avidin sensitive. Carboxylated enzyme is not observed in the presence of 1 mM urea.     

Determination of biocytin

Biocytin (epsilon-N-[d-biotinyl]-L-lysine) is generally undetected in serum and other body fluids of normal healthy individuals in view of the ubiquitous distribution of biotinidase. It has been suggested that biocytin may be present in serum and urine of patients with inherited biotinidase deficiency. We have developed a noncompetitive assay for biocytin based on its interaction with avidin. Biocytin can be determined in biological samples containing both biotin and biocytin. Biotin from such samples is removed by an anion-exchange resin and the biocytin is determined by the avidin-binding assay. The effect of above-normal levels of biotin in the sample on the assay of biocytin is discussed.     

Studies on Production of Biotin by Microorganisms

In a preceding paper we reported that Rhodotorula flava 194 effectively converted biotin to biotinamide. In a present paper the metabolism of desthiobiotin by R. flava 194 was studied under the same condition as in the conversion of biotin to biotinamide. Two desthiobiotin derivatives (Vitamer I and II) were isolated. Vitamer II (crystalline) was identified as bisnordesthiobiotin and Vitamer I was chromatographically determined as desthiobiotinamide.     

Probing the structure of processed antigen by using biotin and avidin. MHC-dependent inhibition of responses to selected biotinyl-insulin derivatives

Current models suggest that Ag undergoes proteolytic cleavage in APC and that resultant peptide fragments associate with class II histocompatibility glycoproteins before recognition by helper T cells. Little direct information is available concerning the physical structure and membrane association of Ag processed under physiologic conditions. A model system, employing a series of biotinylated insulin derivatives, was used to examine the domains of Ag that are presented by APC. We reasoned that avidin should block the response of T cells to a given derivative only if biotin is retained on the functionally relevant form of Ag after processing. By utilizing derivatives modified at selected sites one should be able to determine whether specific sites remain after processing. By using F1 APC pulsed with biotinyl-insulin derivatives modified through the free amino groups of the A1, B1, or B29 amino acids, and T cell hybridomas restricted to I-Ad or I-Ab, we found that avidin inhibited the I-Ad-restricted response to A1, but not B1 or B29 derivatives. By contrast, specific inhibition of the I-Ab-restricted response was observed by using all three derivatives. These results suggest that the processed form of insulin recognized in association with I-Ab is largely intact and includes residues from both chains (A1, B1, and B29). The differential inhibition observed by using T cells restricted to different class II alleles demonstrates that processed Ag associated with I-Ab differs in conformation or structure from that associated with I-Ad. This experimental approach should prove valuable in characterizing the actual structure of processed Ag recognized by T cells.     

A Colorimetric Assay of Lipoyl-N-?-Lysine Hydrolysis Activity Using 2,6-Dibromoquinone-4-Chlorimide

Lipoic acid is a coenzyme for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched chain-ketoacid dehydrogenase, and the glycine cleavage system. Lipoic acid is covalently attached through an amide to the ?-amino group of specific lysine residues of these enzymes. Lipoamidase hydrolyzes the amide bond of lipoyl-N-?-lysine. Because of the difficulty in quantitating lipoic acid or lysine released by hydrolysis of lipoyl-N-?-lysine, a sensitive assay of lipoamidase activity was developed based on quantitation of lipoic acid liberated from lipoyl-?-lysine using 2,6-dibromoquinone-4-chlorimide (DBQC). This method involves acidification of the assay mixture with HCl and separation of lipoic acid from lipoyl-N-?-lysine by extraction into ethyl acetate where it can react with DBQC. This method is as sensitive as methods based on the reaction of lipoic acid with dinitrothiobenzoate and requires only a single extraction, but does not require reduction of the disulfide and the color reagent does not need to be prepared daily. Results obtained using this assay to quantitate lipoic acid released from lipoyl-N-p-aminobenzoate correlated excellently with results obtained using the Marshall–Bratton reaction to quantitatep-aminobenzoate. We have detected lipoyl-N-?-lysine hydrolysis activity that is distinct from that of biotinidase and bile salt-stimulated lipase in lymphoblasts from a patient with biotinidase deficiency. This assay can be used to measure lipoyl-N-?-lysine hydrolysis activity in tissues, especially those with little or no biotinidase activity.     

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.     

Purification of biotinidase from human plasma and its activity on biotinyl peptides

Biotinidase catalyzes the hydrolysis of N epsilon-biotinyllysine (biocytin) to form biotin and free lysine. The enzyme has been purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of (76 +/- 2) X 10(3). The same molecular weight was found by molecular sieve chromatography under nondenaturing conditions, indicating biotinidase is a monomer. This value is in contrast to a molecular weight of 115 000 determined by Pispa [Pispa, J. (1965) Ann. Med. Exp. Biol. Fenn., Suppl. 5, 5-39] with an impure biotinidase. The Km for biocytin was 6.2 X 10(-6) M, and biotinidase was found to be sensitive to phenylmethanesulfonamide and iodoacetamide in agreement with earlier studies by Knappe and co-workers [Knappe, J., Brümmer, W., & Bierderbick, K. (1963) Biochem. Z. 338, 599-613], who suggested that serine hydroxyl groups and sulfhydryl groups are essential for enzymatic activity. The specificity of biotinidase was examined by using synthetic and natural biotinyl peptides isolated by specific proteolytic cleavage of the biotinyl subunit of transcarboxylase. It was found that the rate of hydrolysis of biocytin was 83-fold higher than that found for biotin-containing peptides 5-13 residues in length. Removal of methionine from either side of the conserved region around the biocytin did not greatly alter the rate of cleavage. Increasing the peptide to 65-123 residues in length decreased the rate 1200-fold compared to that of biocytin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigations of the MceIJ-catalyzed posttranslational modification of the microcin E492 C-terminus: linkage of ribosomal and nonribosomal peptides to form "trojan horse" antibiotics

MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide synthetase machinery, to the C-terminal serine residue of microcin E492 (MccE492), an 84 aa ribosomal antibiotic peptide produced by Klebsiella pneumoniae RYC492. The MceIJ-catalyzed formation of the glycosyl ester linkage between MccE492 and the siderophore requires ATP and Mg(II) as cofactors. This work addresses the ATP utilization, mechanism of C-terminal carboxylate activation, and substrate scope of MceIJ. Formation of the ribosomal peptide-nonribosomal peptide linkage between the MccE492 C-terminal decapeptide and monoglycosylated enterobactin (MGE) requires cleavage of the alpha,beta bond of ATP and formation of a putative peptidyl-CO-AMP intermediate. Attack of the peptidyl-CO-AMP carbonyl by the deprotonated C4' hydroxyl of the glucose moiety forms a glycosyl ester linkage with release of AMP. Site-directed mutagenesis of the three cysteines and five histidines in MceI to alanines reveals that these residues are not required structurally or catalytically. MceIJ recognizes all glycosylated enterobactin derivatives formed by the MccE492 gene cluster members MceC ( C-glycosyltransferase) and MceD (esterase) in vitro and a MGE derivative lacking the C6' hydroxyl moiety. The protein complex also accepts and modifies the C-terminal decapeptide substrate fragments of the structurally related microcins H47, I47, and M. MccE492 C-terminal decapeptides bearing fluorescein and biotin moieties on the N-terminus are also substrates for MceIJ, which provides a route for the chemoenzymatic synthesis of enterobactin conjugates with peptide linkages.     

Preparation and characterization of biotinylated psoralen.

Biotinylated psoralen (BPsor), a psoralen derivative containing a biotin moiety attached via a long-chain, positively charged linker, has been synthesized and its interactions with DNA and avidin have been studied. As do other psoralen derivatives, BPsor photoreacts with DNA to form interstrand crosslinks. The biotin binds to streptavidin after the reaction of BPsor with DNA, and this property has been used to measure low levels of BPsor modified DNA by ELISA with streptavidin and biotinylated alkaline phosphatase. In addition, BPsor retains the biological activity of psoralen, as shown by its ability to inhibit lymphocyte proliferation at a level of 10 ng/ml.     

Biotinylated granulocyte/macrophage colony-stimulating factor analogues: effect of linkage chemistry on activity and binding.

Biotinylated granulocyte/macrophage colony-stimulating factor (GM-CSF) analogues with different linkage chemistries and levels of conjugated biotin were synthesized by reacting recombinant human GM-CSF with sulfosuccinimidyl 6-biotinamidohexanoate or biotin hydrazide/1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide. These chemically reactive forms of biotin produced derivatives biotinylated at amine or carboxyl groups, respectively. Amine-derivatized analogues of 1.2 and 3.8 mol of biotin/mol of protein (N1-bGM-CSF and N4-bGM-CSF) and a carboxyl-modified analogue of 4.6 mol of biotin/mol of protein (C5-bGM-CSF) were synthesized. These analogues were compared to determine the effect of biotinylation on biological activity and GM-CSF receptor binding characteristics. The biotinylated proteins migrated with the same molecular weight as the native, unmodified protein as determined by SDS-PAGE and could be detected by Western blotting with alkaline phosphatase conjugated streptavidin, thus demonstrating the biotin linkage. All three analogues retained full agonist activity relative to the native protein (EC50 = 10-15 pM) when assayed for the stimulation of human bone marrow progenitor cell growth. Cell surface GM-CSF receptor binding was characterized by the binding of the analogues to human neutrophils, with detection by fluorescein-conjugated avidin and fluorescence-activated cell sorting. The N-bGM-CSFs demonstrated GM-CSF receptor specific binding that was displaceable by excess underivatized protein, with the detected fluorescence signal decreasing with increasing biotin to protein molar ratio. In contrast, C5-bGM-CSF binding above background fluorescence could not be detected using this system, suggesting that this derivative could bind to and activate the receptor, but not simultaneously bind fluorescein-conjugated avidin. The amine-derivatized biotinylated GM-CSF analogues retained biological activity, could specifically label cell surface receptors, and may be useful nonradioactive probes with which to study GM-CSF receptor cytochemistry and receptor modulation by flow cytometry.     

Role of human serum biotinidase as biotin-binding protein.

Biotinidase shows two binding sites for biotin, with Kd = 59 and 3 nM respectively, and requires tryptophan and cysteine residues of the biotinidase protein for biotin-binding activity. Analysis of human serum by various column-chromatographic techniques indicates that biotinidase is the only protein which exchanges with labelled (+)-biotin. It was shown previously that epileptic patients receiving a high average dose of anticonvulsants (containing a carbamide group) have lower biotin concentrations than those receiving a low dose. We have shown in human serum and with purified biotinidase that these anticonvulsant drugs compete with biotin for binding to the protein moiety.     

Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.