蒽醌及黄酮类化合物与牛血清白蛋白结合的反应研究

用离心超过滤法测定了十四种不同结构的蒽醌及黄酮类化合物与牛血清白蛋白(BSA)的结合常数和结合部位数目,发现这些化合物与BSA 的结合能力随其脂溶性增加而增大,龙胆苦苷不与BSA结合,研究了L-色氨酸,油酸与大黄素对BSA 的竞争结合反应,结果表明L-色氨酸和大黄素拥有一个相同的强结合部位,可以发生1:1 置换反应.低浓度油酸存在下使大黄素等同的6个结合部位区分为两类:n~1=2,n~2=4, 结合部位数目不变,但结合常数显著减小,油酸浓度足够大时,大黄素完全不与BSA结合,测得大黄素与BSA结合的△H≈0.根据上述结果,对蒽醌及黄酮类化合物与BSA结合反应的机理进行了初步探讨.     

Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor.

A protocol based on saturation transfer difference (STD) NMR spectra was developed to characterize the binding interactions at an atom level, termed group epitope mapping (GEM). As an example we chose the well-studied system of galactose binding to the 120-kDa lectin Ricinus communis agglutinin I (RCA(120)). As ligands we used methyl beta-D-galactoside and a biantennary decasaccharide. Analysis of the saturation transfer effects of methyl beta-D-galactoside showed that the H2, H3, and H4 protons are saturated to the highest degree, giving evidence of their close proximity to protons of the RCA(120) lectin. The direct interaction of the lectin with this region of the galactose is in excellent agreement with results obtained from the analysis of the binding specificities of many chemically modified galactose derivatives (Bhattacharyya, L.; Brewer, C. F. Eur. J. Biochem. 1988, 176, 207-212). This new NMR technique can identify the binding epitope of even complex ligands very quickly, which is a great improvement over time-consuming chemical modifications. Efficient GEM benefits from a relatively high off rate of the ligand and a large excess of the ligand over the receptor. Even for a ligand like the biantennary decasaccharide with micromolar binding affinity, the binding epitopes could easily be mapped to the terminal beta-D-Gal-(1-4)-beta-D-GlcNAc (beta-D-GlcNAc = N-acetyl-D-glucosamine) residues located at the nonreducing end of the two carbohydrate chains. The binding contribution of the terminal galactose residue is stronger than those of the penultimate GlcNAc residues. We could show that the GlcNAc residues bind "edge-on" with the region from H2 to H4, making contact with the protein. Analysis of STD NMR experiments performed under competitive conditions proved that the two saccharides studied bind at the same receptor site, thereby ruling out unspecific binding.     

Classification of the binding modes in bovine serum albumin using terminally substituted alkane analogues.

With the fluorescence probe of 8-anilino-1-naphthalenesulfonate (ANS), the binding modes of terminally substituted alkane analogues (C(n)X; X = COOH, OH, CHO, NH(3), CONH(2)) to bovine serum albumin (BSA) were investigated using a competitive binding technique. The Scatchard plot of the fluorometric titration of BSA with ANS showed that the maximum binding number of ANS, n(max), was 3.81, with the binding constant, K(bnd), of 1.42 x 10(6) mol(-1) dm(3). The binding modes of C(n)X to BSA were analyzed based on the fluorometric titration of the ANS and BSA mixture with C(n)X. C(n)COOH completely displaced the ANS bound to BSA, whereas C(n)OH and C(n)CHO displaced only about 40% of the ANS bound to BSA. In contrast, C(n)NH(2) and C(n)CONH(2) displaced very little bound ANS. By comparing these results, we classified the binding modes of C(n)X to BSA into three types. Two of them are detectable with the ANS fluorescence and the remaining one is not detectable with the fluorescence.     

Investigation of the toxic effect of a QDs heterojunction on the interactions between small molecules and plasma proteins by fluorescence and resonance light-scattering spectra

The effect of a ZnO#ZnS QDs heterojunction (O#SQDs) on the binding affinities of flavonoid glycosides for bovine serum albumin (BSA) was investigated. The fluorescence intensities of BSA decreased remarkably with increasing concentration of O#SQDs. The magnitudes of the binding constants of flavonoid glycosides for BSA in the presence of O#SQDs were in the range of 10(5)-10(7) L mol(-1), and the number of binding sites per BSA (n) was determined as 1.24 ± 0.17. O#SQDs increased the affinities of flavonoid glycosides for BSA by about 2.96% to 114.68% depending on their structures. O#SQDs in blood will enhance the transportation of flavonoid glycosidegs in blood and improve their pharmacology effects. From this point, O#SQDs are a perfect candidate for flavonoid glycosides delivery applications.     

Spectroscopic studies on binding of 1-phenyl-3-(coumarin-6-yl)sulfonylurea to bovine serum albumin

The interaction of 1-phenyl-3-(coumarin-6-yl)sulfonylurea (SU22) with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectroscopy combined with UV-absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy techniques under simulative physiological conditions for the first time. Fluorescence data and UV-absorption spectra revealed that the quenching mechanism of fluorescence of BSA by SU22 was a static quenching process and the number of binding sites was about 0.8858; the thermodynamic parameters (DeltaG=-29.23 kJ mol(-1), DeltaH=-47.48 kJ mol(-1), and DeltaS=-61.24 J mol(-1)K(-1)) explained that hydrogen bond and Van der Waals interaction were the main binding force stabilizing the complex. The binding average distance between SU22 and BSA was obtained (3.20 nm) on the basis of the F?rster's theory. In addition, The CD spectra and FT-IR spectra have proved that BSA secondary structure changed in the presence of SU22 in aqueous solution.     

J-aggregation of anionic ethyl meso-thiacarbocyanine dyes induced by binding to proteins

The effects of ribonuclease A (RNase), lysozyme, trypsin, and bovine serum albumin (BSA) on the J-aggregation behavior of 3,3'-bis[sulfopropyl]-5-methoxy-4',5'-benzo-9-ethylthiacarbocyanine (1), 3,3'-bis[sulfopropyl]-4,5,4',5'-dibenzo-9-ethylthiacarbocyanine (2), and 3,3'-bis[sulfopropyl]-5,5'-dimethoxy-9-ethylthiacarbocyanine (3) were studied in aqueous solution. The formation of J-aggregates at pH 6 is induced by RNase for 1-3, by lysozyme for 1 and 2, and by trypsin for 2. The formation of J-aggregates correlates with decay of the dimers and is supported by induced circular dichroism spectra. The concentration of J-aggregates for lysozyme/1 increases with an increase in the protein/dye concentration ratio, reaches a plateau, and then gradually decreases. J-aggregates are characterized by relatively weak fluorescence; e.g., Phi(f) = 0.01 for lysozyme/1, and by a small Stokes shift of 6-8 nm, indicating almost resonance fluorescence. J-aggregation proceeds in the range of seconds to minutes with sigmoidal type kinetic curves for trypsin/2 and nonsigmoidal kinetics in the other cases. The presence of BSA, in contrast to RNase, lysozyme, and trypsin, results in deaggregation of dimers of 1-3 and formation of bound monomers and exhibits intense fluorescence from the trans-monomer; e.g., Phi(f) = 0.22 for BSA/1. Generally, the binding of 1-3 to the proteins is a cooperative process, where the number of binding sites changes from n = 15 for lysozyme/1 to n = 6 for trypsin/2 and n = 0.3 and 1 for BSA/3.     

Incorporation of a monolithic column into sequential injection system for drug-protein binding studies

A sequential injection analysis (SIA) manifold was incorporated with a monolithic strong anion-exchanger disk for on-line drug-protein interaction studies. The antibiotic ciprofloxacin (CF) was selected as a model drug compound. The separation principle was based on the strong retention of bovine serum albumin (BSA) on the monolithic strong anion-exchanger and the liberation/release of the free form of the drug. Elution of the retained BSA was easily achieved by delivering a different mobile phase via the SIA manifold. The type of functional group of the monolithic support, the breakthrough volume and the injected volumes of CF and BSA were studied and optimized. The influence of the variation of incubation time was studied in on-line binding assays. Scatchard plot was employed to obtain the number of binding sites and the equilibrium binding constants. For the off-line study of the CF-BSA binding, two binding classes were determined with constants of (3.16+/-0.21)x10(6)M(-1) and (1.27+/-0.48)x10(4)M(-1) and 6.1+/-1.3 and 17.8+/-3.9 binding sites per class, respectively. In non-equilibrium binding experiments the binding rate constant was k(1)=785 M(-1)min(-1). All measurements were monitored with fluorescence (lambda(ext)=300 nm, lambda(em)=460 nm) and spectrophotometric detection (lambda=280 nm). To evaluate the accuracy of the developed method the obtained results were compared versus ultrafiltration experiments and were found in good agreement.     

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions

In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).     

Carbohydrate-protein interactions at interfaces: synthesis of thiolactosyl glycolipids and design of a working model for surface plasmon resonance

Thiolactosyl lipids designed for carbohydrate-protein binding studies have been synthesised. One representative was selected for binding studies with a plant lectin RCA120, the agglutinin from Ricinus communis. The interactions were measured quantitatively in real time using a BIAcore surface plasmon resonance instrument. Removal of much of the galactose from the thiolactosyl lipid in situ with beta-galactosidase showed that the lectin binding was highly specific. A dissociation constant KD = 8.77 x 10(-8) M was measured for 1-[2-[2-(2-[beta-D-galactopyranosyl-(1-->4)-1-thio-beta-D -glucopyranosyl]ethoxy)ethoxy]ethoxy]octadecane 30 which is four orders of magnitude greater than that determined for binding to lactose in solution. A concentration of lactose of > 80 mM was required to block the lectin binding to thiolactosyl lipid in a neomembrane.     

荧光光谱法研究除草醚与牛血清白蛋白的相互作用

运用荧光光谱和紫外吸收光谱研究水溶液中除草醚(NP)与牛血清白蛋白(BSA)的相互作用.结果表明,NP与BSA形成基态复合物导致BSA内源荧光猝灭,猝灭机理主要为静态猝灭和非辐射能量转移.运用位点模型计算298 K、308 K、318 K时结合常数K_A分别为6.97×10~4、5.25×10~4 、4.96×10~4 L·mol~(-1),结合位点数n分别为0.98、0.92、0.96.根据热力学参数确定其作用力以疏水作用和静电作用为主;运用F(o)rster偶极-偶极非辐射能量转移原理,测定了NP与BSA的结合距离r为2.19 nm;用同步荧光技术初步考察了NP对BSA构象的影响.     

膳食黄酮类化合物与牛血清蛋白的分子结构亲和力关系

用荧光滴定法研究了食用黄酮类化合物的分子结构及其与牛血清蛋白间的亲和力关系。研究证明食用黄酮类化合物与牛血清蛋白的结合过程在很大程度上受黄酮结构的影响,类黄酮中羟基的甲基化使其与牛血清蛋白的亲和力增长1～794倍。环A、B、C的羟基化作用对类黄酮与牛血清蛋白的亲和力也有很大影响,糖基化作用因共轭位置和糖种类的不同而使类黄酮与牛血清蛋白的结合力降低1～2个数量级。C2‖C3双键的加氢化作用使结合能力稍有减弱。与非聚合型儿茶酚和邻苯二酚型儿茶酚相比,聚合儿茶酚和邻苯三酚型儿茶酚与牛血清蛋白的亲和力更强。分配系数升高时,类黄酮与牛血清蛋白的亲和力增强;氢键的电子供体和受体数目增多时,类黄酮与牛血清蛋白的亲和力降低,表明疏水力是影响其相互结合作用的主要因素。     

Binding position of phenylbutazone with bovine serum albumin determined by measuring nuclear magnetic resonance relaxation time

The binding of phenylbutazone (PB) to bovine serum albumin (BSA) was considered to be predominantly due to hydrophobic interaction based on the thermodynamic parameters obtained by an equilibrium dialysis method. Little variation of proton nuclear magnetic resonance (1H-NMR) chemical shift of PB was found with change in the concentration of PB (0.5-5 mM) or upon the addition of BSA (7.25 x 10(-5) M). The NMR spectrum of PB in 0.1 M phosphate buffer solution at pH 7 showed that PB existed as a mesomeric anion. The spin-lattice relaxation time (T1) of PB was almost concentration-independent, but decreased in the presence of BSA to 36-38% for the phenyl group and 48-100% for the butyl group. The spin-spin relaxation time (T2) of PB was also almost independent of concentration, but was remarkably decreased in the presence of BSA to ca. 2.5% for the phenyl group and ca. 6-9% for the butyl group. The ratios of the spin-spin relaxation rate (1/T2) of the free PB to that of the bound PB were ca. 5000-11000 for the butyl group and ca. 23000 for the phenyl group. The binding of PB to BSA was considered to involve primarily the phenyl group.     

Effects of acetonitrile on adsorption behavior of bovine serum albumin onto synthetic calcium hydroxyapatite particles

The objective of this study was to examine the effects of acetonitrile (AN) on the adsorption behavior of bovine serum albumin (BSA) onto calcium hydroxyapatite [Ca10(PO4)6(OH)2 Ca10, Hap] materials by combining the ultraviolet (UV) and circular dichroism (CD) measurements of BSA solution. The structural change of BSA molecules with addition of AN was investigated by UV and CD spectroscopy measurements prior to studying adsorption behavior of BSA onto Hap. The CD spectra revealed that the fraction of alpha-helical content of BSA is remarkably decreased at AN concentrations above 30 vol.%, while beta-sheet content is increased. On the other hand, the percentages of random coil and turn contents were decreased only slightly. In addition to this secondary structural change of BSA, the UV spectra suggested that the tertiary structure of protein molecules was also changed by the addition of large amounts of AN; BSA molecules associate to form molecular aggregates at [AN]> or =40 vol.%. From the adsorption of BSA onto Hap particles (ca. 30 nm in the particle length) from a water-AN mixed solution, it was revealed that the adsorption behavior of BSA strongly depends on the change of secondary and tertiary structures of BSA by addition of AN. The contraction of BSA molecules at low AN concentrations (10-20 vol.%) gave their small cross-sectional area, providing a large amount of adsorption (n(BSA)), although n(BSA) was decreased above 30 vol.% AN by enlargement of BSA molecules with solvation and unfolding some alpha-helix domains. The n(BSA) values of the systems with AN exhibited a maximum; n(BSA) was increased at a lower BSA concentration region, although it was decreased at a higher BSA concentration due to self-association. Accompanying the change of n(BSA) with AN addition, the maxima of electrophoretic mobility (em) of the Hap particles were observed for the systems with AN, although the em of Hap particles was normally increased and saturated with increase in protein coverage for the native structure on the system without AN. On the other hand, because the aggregated BSA molecules could be cooperatively bound, the adsorption of BSA onto the Hap particles with large size (108 nm in the particle length) was enhanced in the presence of AN.     

Efficacy of glycoprotein enrichment by microscale lectin affinity chromatography

Reproducible and efficient affinity enrichment is increasingly viewed as an essential step in many investigations leading to the discovery of new biomarkers. In this work, we have evaluated the repeatability of lectin enrichment of glycoproteins from human blood serum through both qualitative and quantitative proteomic approaches. In a comprehensive evaluation of lectin binding, we have performed 30 separate microscale lectin affinity chromatography experiments, followed by a conventional sample purification, and LC-MS/MS analysis of the enriched glycoproteins. Two lectin affinity matrixes, both with Con A lectin, immobilized to the same solid support but differing in the amount of immobilized lectin, were investigated to characterize their binding properties. Both qualitative and quantitative data indicate acceptable repeatability and binding efficiency for the lectin materials received from two different commercial sources.     

Protein adsorption characteristics of calcium hydroxyapatites modified with pyrophosphoric acids

Protein adsorption characteristics of calcium hydroxyapatite (Hap) modified with pyrophosphoric acids (PP(a)) were examined. The PP(a) modified Hap particles (abbreviated as PP-Hap) possessed anchored polyphosphate (PP: P-{O-PO(OH)}(n)-OH) branches on their surfaces. The proteins of bovine serum albumin (BSA: isoelectric point (iep)=4.7, molecular mass (M(s))=67,200 Da, acidic protein), myoglobin (MGB: iep=7.0, M(s)=17,800 Da, neutral protein), and lysozyme (LSZ: iep=11.1, M(s)=14,600 Da, basic protein) were examined. The zeta potential (zp) of PP-Hap particles as a function of pH overlapped; zp-pH curves were independent of the concentration of pyrophosphoric acids (abbreviated as [PP(a)]) used for modifying Hap surface. The saturated amounts of adsorbed BSA (Delta n(ads)(BSA)) were increased three-fold by the surface modification with PP(a) though they were independent of the [PP(a)]. Furthermore, the fraction of BSA desorption was independent of the [PP(a)]. This enhancement of BSA adsorption onto the PP-Hap is due to the hydrogen bonding between oxygen and OH groups of the PP-branches and functional groups of BSA molecules. In the case of LSZ, a more higher adsorption enhancement was observed; the saturated amount of adsorbed LSZ (Delta n(ads)(LSZ)) for Hap modified at [PP(a)]=6 mmol/dm(3) was nine-fold than that for Hap unmodified. This remarkable adsorption enhancement was explained by a three-dimensional binding mechanism; LSZ molecules were trapped inside of the PP-branches. Hence, a fraction of LSZ desorption was decreased with an increase in the [PP(a)]; as more PP-branches are presented on the surface the higher retardation of LSZ desorption was induced. It was expected from their small size that MGB adsorb between the PP-branches as well as LSZ. However, the amounts of adsorbed MGB (Delta n(ads)(MGB)) did not vary and were independent of the [PP(a)] due to the small numbers of functional groups of MGB. In addition, no dependence of the fraction of MGB desorption on the [PP(a)] was observed. The results of zp for all the protein systems supported the mode of protein adsorption discussed. The anchored structure of the PP-branches developed on the Hap surface to provide three-dimensional protein adsorption spaces was proved by a comparative experiment that was elucidating the effect of pyrophosphate ions for BSA adsorption onto Hap.     

Antioxidant activity of bovine serum albumin binding amino acid Schiff-bases metal complexes

Glutamic acid-salicylaldehyde Schiff-base metal complexes are bound into bovine serum albumin (BSA),which afforded BSA binding Schiff-base metal complexes (BSA-SalGluM,M=Cu,Co,Ni,Zn).The BSA binding metal complexes were characterized by UV-vis spectra and Native PAGE.It showed that the protein structures of BSA kept after coordinating amino acid Schiff-bases metal complexes.The effect of the antioxidant activity was investigated.The results indicate that the antioxidant capacity of BSA increased more than 10 times after binding Schiff-base metal complexes.     

Structure and function of complement activating enzyme complexes: C1 and MBL-MASPs

The complement system is a major effector arm of the immune defense contributing to the destruction of invading pathogens. There are three possible routes of complement cascade activation: the classical, the alternative and the lectin pathways. The activation of the classical and lectin pathways is initiated by supramolecular complexes, which resemble each other. Each complex has a recognition subunit (C1q in the classical and mannose-binding lectin (MBL) in the lectin pathway), which associates with serine protease zymogens (C1q with C1r and C1s, and MBL with MBL-associated serine proteases: MASP-1, MASP-2) to form the C1 and MBL-MASPs complexes, respectively. As the recognition subunits bind to activator structures, subsequent activation of the serine protease zymogens occurs. The precise structure of the complexes and the exact mechanism of their activation have not been solved, yet. In this review we summarize the recent advances about the structure and function of the individual subcomponents of both complexes achieved by genetic engineering, molecular modeling, physico-chemical and functional studies. Special emphasis will be laid on the serine proteases: the role of the individual domains in the assembly of the C1s-C1r-C1r-C1s tetramer and in the control of the protease activity will be discussed. We will then focus on recent functional models of the supramolecular complexes. The question of how a non-enzymatic signal (the binding of C1q or MBL to activators) can be converted into enzymatic events (activation of serine protease zymogens) will be addressed. The similarities and differences between C1 and MBL-MASPs will also be discussed.     

T(4-Mop)PS4卟啉与牛血清蛋白相互作用的研究

以四-(4-甲氧基-3-磺酸基苯)卟啉(T(4-Mop)PS4)为探针,通过T(4-Mop)PS4与牛血清白蛋白(BSA)的相互作用,建立了测定BSA的电化学分析方法。T(4-Mop)PS4的峰电流变化(ΔIp)与BSA在2.0×10-6～1.0×10-5mol.L-1范围内呈良好的线性关系,检出限为1.2×10-6mol.L-1;对5.0×10-6mol.L-1BSA平行测定8次,其相对标准偏差为2.0%,回收率为95%～104%。组氨酸、缬氨酸、苯丙氨酸、丝氨酸、异白氨酸、谷氨酰胺、苏氨酸等氨基酸对BSA的测定不产生干扰。采用紫外可见光度法、荧光光谱法和线性扫描伏安法(LSV)研究了T(4-Mop)PS4与BSA之间的相互作用,并测定了二者相互作用的结合常数和结合比。研究表明,T(4-Mop)PS4与BSA之间主要以疏水作用力结合,形成了1∶1的稳定复合物。     

Profiling of Endo H-released serum N-glycans using CE-LIF and MALDI-TOF-MS--application to rheumatoid arthritis

High-mannose and hybrid-type N-glycans are present in human serum glycoproteins in low abundance but have recently been described to play an important role in immune responses. It is therefore important to find a strategy to selectively analyze their structures in the context of health and disease in order to understand their impact on disease mechanisms. We report here the characterization of high-mannose and hybrid-type N-glycans in total human serum. To this end, N-glycans were released using Endo-β-N-acetylglucosaminidase H (Endo H) and analyzed by CE-LIF and MALDI-TOF-MS. We found that the high-mannose structures Man(5-9)GlcNAc(1) represented the majority of the pool. The monoglucosylated structure Glc(1)Man(9)GlcNAc(1) as well as four hybrid structures could be identified. Then, we compared the Endo H-released serum glycome of patients suffering from rheumatoid arthritis with healthy controls as mannose-binding lectin deficiency (MBL) and modulation of α-mannosidase activity were previously associated with this disease. Interestingly, we observed that both high-mannose and hybrid structures were fairly constant, suggesting that circulating MBL and α-mannosidase may not affect significantly the levels of serum glycoproteins carrying these glycans.     

Fluorescence Anisotropy Assays Reveal Affinities of C- and O-Glycosides for Concanavalin A(1)

The free energies of binding of various C- and O-glycosides to the lectin concanavalin A were measured using fluorescence anisotropy. Fluorescein derivatives of mannose and glucose were synthesized and were shown to bind to concanavalin A with free energies of -5.1 and -4.3 kcal mol(-)(1), respectively. Competition experiments were performed to determine the binding energies of different nonfluorescent carbohydrates, and the results were in excellent agreement with the binding energies determined by microcalorimetry. The minimum carbohydrate epitope that fills the lectin carbohydrate binding site, methyl 3,6-di-O-(alpha-mannopyranosyl)-alpha-mannopyranoside, competes directly for the site with the fluorescent ligands, indicating that the fluorescent ligands bind specifically. The binding affinities of C-glycosides to concanavalin A were compared with those of O-glycosides. The free energies of binding for corresponding C- and O-glycosides differed by less than 0.5 kcal mol(-)(1), indicating that recognition properties of C- and O-glycosides are very similar. It was found that for some ligands the use of a carbon linkage rather than an oxygen linkage caused the specificity of binding to decrease slightly.