Average Dissociation Constant Research Articles

The Effect of Monovalent Cations on the Thermal Stability of Small DNA Oligomers with Internal Loops

Although the effect of monovalent cations on the thermal stability of nucleic acid duplexes has been studied for many years, relatively little is known about cation effects on the stability of DNAs or RNAs with internal loops. We have therefore investigated the thermal stability of the DNA analog of the let-7 microRNA::lin-41 messenger RNA complex from C. elegans, which contains an asymmetric internal loop that kinks the helix backbone1. A DNA construct containing a symmetric internal loop at the same site was also studied, as well as a fully base-paired control. The melting temperatures of the two oligomers with internal loops are equal at low ionic strengths in solutions containing Na+ ions. However, the melting temperature of the oligomer with the asymmetric loop is ∼2°C higher in solutions with Na+ ion concentrations greater than ∼100 mM. The melting temperature of the oligomer with the asymmetric loop is also ∼2°C higher than the oligomer with the symmetric loop when the solution contains K+ ions. Binding studies indicate that both oligomers with internal loops, as well as the duplex control, bind Na+ ions weakly, with an average dissociation constant of 150 ± 10 mM. Hence, high concentrations of Na+ ions appear to stabilize DNAs with asymmetric internal loops, most likely because of electrostatic screening of the closely spaced phosphate groups near the kink site. Surprisingly, none of the oligomers bind K+ ions over the concentration range tested. Supported in part by the Analytical and Surface Chemistry Program of the National Science Foundation. 1 M. Cevec, C. Thibaudeau, J. Plavec (2008) Nucleic Acids. Res. 36, 2330-2337.

Hybrid lactoferrins containing transferrin C1 or C2 subdomains bind asialoglycoprotein receptors

The Fe‐binding proteins lactoferrin (Lf) and transferrin (Tf) are structurally similar, each Fe‐binding lobe (N, C) composed of two sub‐domains (N1/N2; C1/C2). Lf, but not Tf, binds the major subunit of the asialoglycoprotein receptor (RHL1) in a galactose‐independent manner. To identify C‐lobe regions involved in RHL1 binding, we generated recombinant Lf‐Tf hybrid proteins in which Tf C1 or C2 subdomains were positioned within a Lf background. cDNAs of human Lf and Tf encoding Lf:TfC1 and Lf:TfC2 were constructed and expressed in Sf9 insect cells. Binding constants of purified native and recombinant Lfs for RHL1 were determined by surface plasmon resonance. Binding of desialylated ligands and native and recombinant Lf proteins to immobilized RHL1 required Ca2+ and neutral pH. Tf did not bind RHL1 under any conditions. Average dissociation constants measured for the various proteins were as follows: native Lf: 138 ± 44 nM; r‐Lf: 87 ± 50 nM; Lf:TfC1: 31 ± 28 nM; Lf:TfC2: 150 ± 76 nM; and asialofetuin: 21 ± 16 nM. Binding constants for native and recombinant Lfs did not differ statistically. Thus, Tf C1 or C2 sub‐domains in a Lf background showed no loss of RHL1‐binding activity. We conclude that the RHL1 binding determinants on Lf do not reside solely in one of the C‐lobe subdomains. (Support: Research Corporation, NIH DK‐61984).

Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1

Transferrin receptor 1 (R(D)) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO(2)(2+)). Can the uranyl-saturated transferrin (TUr(2)) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr(2) interacts with R(D) in two different steps. The first is fast, direct rate constant, k(1) = (5.2 +/- 0.8) x 10(6) M(-1) s(-1); reverse rate constant, k(-1) = 95 +/- 5 s(-1); and dissociation constant K(1) = 18 +/- 6 microM. The second occurs in the 100-s range and leads to an increase in the stability of the protein-protein adduct, with an average overall dissociation constant K(d) = 6 +/- 2 microM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr(2) and the C-lobe of iron-loaded transferrin toward the interaction with R(D).

A quantitative immunosensing technique based on the measurement of nanobeads’ Brownian motion

A novel bio-sensing technique based on the measurement of nanobeads’ Brownian motion using a micro-particle tracking velocimetry (micro-PTV) has been successfully developed to detect antigen–antibody interactions. The rapid interaction between antigens (C-reactive proteins, CRPs) and nanobeads with conjugated antibodies (anti-CRPs) has enabled real-time detection of CRPs to be easily carried out. During the binding process of CRPs to nanobeads, the mean value of the beads’ diameters increases so that the Brownian velocity decreases with the increase of time. Moreover, higher CRP concentration leads to a lower Brownian velocity of the nanobeads in the equilibrium state. From the results, the limit of detection 0.1μg/ml was observed and could be further improved by using smaller nanobeads. Moreover, based on kinetic analysis, the average dissociation constant KD=(6.48±1.43)×10−7 found by this technique for anti-CRP was shown to be in close agreement with the literature values obtained by dual-polarization interferometry (DPI) and indirect competition enzyme-linked immunosorbent assay (ELISA). This simple sensing method can further be used to detect other bio-molecules and viruses.

The carbohydrate-binding domain on galectin-1 is more extensive for a complex glycan than for simple saccharides: implications for galectin–glycan interactions at the cell surface

gal-1 (galectin-1) mediates cell–cell and cell–extracellular matrix adhesion, essentially by interacting with β-galactoside-containing glycans of cell-surface glycoconjugates. Although most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyl-lactosamine, this view is limited, because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. In the present study we used NMR spectroscopy to investigate the interaction of human gal-1 with a large (120 kDa) complex glycan, GRG (galactorhamnogalacturonate glycan), that contains non-randomly distributed mostly terminal β(1→4)-linked galactose side chains. We used 15N–1H-HSQC (heteronuclear single quantum coherence) NMR experiments with 15N-enriched gal-1 to identify the GRG-binding region on gal-1 and found that this region covers a large surface area on gal-1 that includes the quintessential lactose-binding site and runs from that site through a broad valley or cleft towards the dimer interface. HSQC and pulsed-field-gradient NMR diffusion experiments also show that gal-1 binds GRG with a gal-1:GRG stoichiometry of about 5:1 (or 6:1) and with average macroscopic and microscopic equilibrium dissociation constants (Kd) of 8×10−6 M and 40×10−6 M (or 48×10−6 M) respectively, indicating stronger binding than to lactose (Kd=520×10−6 M). Although gal-1 may bind GRG in various ways, the glycan can be competed for by lactose, suggesting that there is one major mode of interaction. Furthermore, even though terminal motifs on GRG are Gal-β(1→4)-Gal rather than the traditional Gal-β(1→4)-Glc/GlcNAc (where GlcNAc is N-acetylglucosamine), we show that the disaccharide Gal-β(1→4)-Gal can bind gal-1 at the lactose-binding domain. In addition, gal-1 binding to GRG disrupts inter-glycan interactions and decreases glycan-mediated solution viscosity, a glycan decongestion effect that may help explain why gal-1 promotes membrane fluidity and lateral diffusion of glycoconjugates within cell membranes. Overall, our results provide an insight into the function of galectin in situ and have potential significant biological consequences.

Cobalt and the Iron Acquisition Pathway: Competition towards Interaction with Receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO 3 2 − to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co 3 + and CO 3 2 − from CoNtaCO 3 2 − to the metal-binding site in the C-lobe of T: direct rate constant, k 1 = (1.1 ± 0.1) × 10 6 M − 1 s − 1 ; reverse rate constant, k − 1 = (1.9 ± 0.6) × 10 6 M − 1 s − 1 ; and equilibrium constant, K = 1.7 ± 0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k 2 = (3 ± 0.3) × 10 6 M − 1 s − 1 ; reverse rate constant, k − 2 = (1.6 ± 0.3) × 10 − 2 s − 1 ; and equilibrium constant, K 2a = 5.3 ± 1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo 2) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo 2 with the helical domain of R1: direct rate constant, k 3 = (4.4 ± 0.6) × 10 10 M − 1 s − 1 ; reverse rate constant, k − 3 = (3.6 ± 0.6) × 10 4 s − 1 ; and dissociation constant, K 1d = 0.82 ± 0.25 μM. The second is a very slow interaction of the N-lobe of TCo 2 with the protease-like domain of R1. This increases the stability of the protein–protein adduct by 30-fold with an average overall dissociation constant K d = 25 ± 10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE—with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe—competes with iron-saturated transferrin in an unknown state towards interaction with R1.

Oxygen-dependent Oxidation of Fe(II) to Fe(III) and Interaction of Fe(III) with Bovine Serum Albumin, Leading to a Hysteretic Effect on the Fluorescence of Bovine Serum Albumin

The serum albumin is the most abundant protein in blood plasma and the iron is essential for many cellular processes. However, the interaction between Fe(3+) and haem-free serum albumin remains unclear. Here we provide evidence for the fact that haem-free BSA possesses one specific Fe(3+)-binding site. The binding of Fe(3+) to BSA results in a significant quenching of the Trp fluorescence of BSA. The average apparent dissociation constant value for the interaction of Fe(3+) and BSA is 3.46 x 10(-8)+/-3 x 10(-10) M at 37 degrees C and 3.30 x 10(-8)+/-5 x 10(-10) M at 25 degrees C, respectively, as determined by fluorescence titration. Addition of 50 microM Fe(2+) to 1 microM BSA results in an obvious hysteretic effect on the fluorescence of BSA. The time-dependent fluorescence quenching of BSA by Fe(2+) is not caused by the Fe(2+)-induced conformational change of BSA, but the oxygen-dependent oxidation of Fe(2+) to Fe(3+). Fe(2+) undergoes an oxygen-dependent oxidation to Fe(3+) under aerobic conditions, which is accelerated by the interaction of BSA with Fe(3+) and extensively inhibited under anaerobic conditions. The results suggest that BSA may take part in non-transferrin bound iron transfer.

Unexpected relationship between plasma protein binding and the pharmacodynamics of 2‐NAP, a CCK1‐receptor antagonist

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT?: * Two chemically diverse CCK1 receptor antagonists have been shown clinically to inhibit CCK-evoked contraction of human gallbladder [2, 3]. These studies have not examined the relationship between plasma concentration and effect, the latter usually considered to be predictive from the free drug concentration [8]. * We wanted to examine our novel CCK1 receptor antagonist in this validated model and also to explore its PK-PD relationship. * 2-NAP inhibited CCK-evoked human gallbladder contraction in vivo but at a plasma free concentration that was, in theory, too low to have achieved adequate CCK1 receptor occupancy. * The study serves as a caveat to the assumption that free plasma concentration can be used to predict pharmacological effect. To study the pharmacokinetics and pharmacodynamics of 2-NAP (2-naphthalenesulfonyl-L-aspartyl-(2-phenethyl)amide), a selective CCK1 receptor antagonist in healthy volunteers. 2-NAP was given to 12 healthy male volunteers in an ascending dose, safety and PK phase 1a study by 1 h i.v. infusion (0.6-9.6 mg kg(-1) h(-1)). A further 12 healthy male volunteers received i.v. CCK-8S (6.25 pmol kg(-1) h(-1)) to produce gallbladder contraction, measured by ultrasound recordings of gallbladder volume, and the effect of concurrent i.v. 2-NAP administration was studied. Plasma protein binding in vitro and ex vivo was measured by ultrafiltration and by equilibrium dialysis. 2-NAP was generally well tolerated, displayed linear pharmacokinetics and a very high degree of plasma protein binding (99.9%). A 105 min i.v. CCK-8S infusion induced a reduction in gallbladder volume of 14.9 (+/-7.0) ml during placebo co-infusion and this was reduced to 2.4 (+/-5.9) ml when 2-NAP was co-infused with CCK-8S (P = 0.00024, paired t-test, mean change 12.5 ml; 95% CI For mean 7.4, 18.3 ml). This extent of inhibition was consistent with a 2-NAP total plasma concentration of 36 microm, but when protein binding corrections were made, the 'free concentration' of 2-NAP was only 0.04 microm, a value much less than the average equilibrium dissociation constant of 2-NAP for human CCK1 receptors ( approximately 0.7 microm). The pharmacological effect of a drug is usually considered to be determined by its free concentration. However, the complete inhibition of CCK-8S-evoked gallbladder contraction by a free plasma concentration of 0.04 microm 2-NAP was much greater than would have been predicted from simple drug-receptor occupancy theory and cautions against the general use of free concentration of drug for predicting pharmacological effect.

Syntheses and in vitro evaluation of fluorinated naphthoxazines as dopamine D2/D3 receptor agonists: radiosynthesis, ex vivo biodistribution and autoradiography of [18F]F-PHNO

Carbon-11-labeled (+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ([(11)C]-(+)-PHNO) is a dopamine D2/D3 agonist radioligand that is currently used to image the high-affinity state of dopamine receptors in humans with positron emission tomography (PET). The present study reports the preparation and evaluation of fluorinated (+)-PHNO derivatives. Five fluorinated (+)-PHNO derivatives were synthesized and tested in vitro for inhibition of binding of [(3)H]domperidone in homogenates of rat striatum and inhibition of binding to [(3)H]-(+)-PHNO in homogenates of human-cloned D2Long receptors in Chinese hamster ovary cells and rat striatum. Radiolabeling with fluorine-18 was carried out for the most promising candidate, N-fluoropropyl-(+)-HNO (F-PHNO), and ex vivo biodistribution and autoradiography studies with this radiopharmaceutical were performed in rodents. (+)-PHNO and the fluorinated analogs inhibited binding of [(3)H]domperidone and [(3)H]-(+)-PHNO to the high- and low-affinity states of dopamine D2 receptors, consistent with D2 agonist behavior. The average dissociation constant at the high-affinity state of D2, K(i)(High), was 0.4 nM for F-PHNO and proved to be equipotent with (+)-PHNO (0.7 nM). All other fluorinated derivatives were significantly less potent (K(i)(High)=2-102 nM). The most promising candidate, F-PHNO, was labeled with fluorine-18 in 5% uncorrected radiochemical yield, with respect to starting fluoride. Ex vivo biodistribution and autoradiography studies in rodents revealed that [(18)F]F-PHNO rapidly enters the rodent brain. However, this radiotracer does not reveal specific binding in the brain and is rapidly cleared. Five novel dopamine D2/D3 agonists based on (+)-PHNO were synthesized and evaluated in vitro. F-PHNO was shown to behave as a potent D2 agonist in vitro and was therefore radiolabeled with fluorine-18. Despite the promising in vitro pharmacological profile, [(18)F]F-PHNO did not display in vivo behavior suitable to image dopaminergic receptor expression using PET.

Metal-Linked Dimerization in the Iron-Dependent Regulator from Mycobacterium tuberculosis

The iron-dependent regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. The natural ligand for IdeR is Fe(II), but Ni(II), Co(II), Cd(II), Mn(II), and Zn(II) also bind to and activate the protein in vitro. Protein activation by metal is a complex process involving metal-induced folding of the N-terminal domain, changes in the interaction between the N- and C-terminal domains, and the formation of homodimers. Here, we investigate the energetics of dimerization and metal binding in IdeR. The dimerization energetics were determined as a function of metal binding using equilibrium analytical ultracentrifugation. The equilibrium dimer dissociation constant of apo-IdeR was 4.0 microM at 20 degrees C. The dissociation constant decreased to 0.5 microM in the presence of one equivalent of Ni(II)Cl(2) and decreased further (K(d) << 50 nM) in the presence of excess Ni(II). IdeR contains two tryptophan residues. The addition of Ni(II) induced changes in fluorescence intensity and emission maximum of the tryptophan residues that strongly depended on protein concentration. At low IdeR concentration, fluorescence was enhanced at low metal-to-protein ratios but was quenched at high metal-to-protein ratios. At high IdeR concentration, metal binding resulted only in fluorescence quenching. The fluorescence enhancement at low protein concentrations was buffer-dependent and required the presence of both tryptophans. Metal binding affinity was measured quantitatively using equilibrium dialysis. The results showed strongly positive cooperative binding of three equivalents of metal per monomer with an average apparent dissociation constant of 2.2 +/- 0.3 microM and a Hill coefficient of 2. Metal binding was not cooperative in an IdeR variant that showed reduced affinity for dimer formation. The results of this study establish the positive cooperative nature of metal binding by IdeR and suggest that dimerization is a major contributor to cooperative binding. The strong coupling between metal binding and dimerization places specific constraints on the activation mechanism.

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC 50 for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC 50 for inhibiting formation of the soluble C3 convertase (5 nM) is 50–80-fold below the normal serum concentration of C4BP (250–400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.

Truncated Semenogelin I Binds Zinc and Is Cleaved by Prostate‐Specific Antigen

Semenogelins I and II are major coagulum-forming proteins in semen, and they are secreted mainly by the seminal vesicles. These proteins bind Zn(2+) and act as substrates for prostate-specific antigen and transglutaminase. A variant semenogelin I lacking 60 amino acids has been described that occurs in different populations with an allele frequency of 1%-3%. To better understand the function of the semenogelins in vivo, our aim was to characterize the properties of the variant form and compare with the wild type. Recombinant proteins were synthesized in insect cells. Binding of Zn(2+) was studied by titration of metal ions in the presence of a zinc (II) fluorophore chelator. SDS-PAGE was used to visualize the results of cleavage by prostate-specific antigen and cross-linking with transglutaminase. We found that the truncated and wild-type semenogelin molecules had similar Zn(2+)-binding properties (ie, a stoichiometry of at least 9-10 mol per mol of protein and an average dissociation constant of 5 mumol/L per site), and they showed also similar susceptibility for degradation by prostate-specific antigen. Furthermore, like the wild-type form, the truncated semenogelin I was able to serve as a substrate for transglutaminase. These findings imply that the studied characteristics do not depend on a well-defined tertiary structure, or that the deletion has no major effect on the structure responsible for these features.

A novel specific heparin‐binding activity of bovine folate‐binding protein characterized by capillary electrophoresis

Folate-binding proteins (FBPs) are ubiquitous, soluble and membrane-bound high-affinity receptors for folate, an essential nutrient involved in nucleic and amino acid metabolism. In the course of optimizing CE separation conditions for FBP purified from cow's milk we discovered a novel specific heparin-binding activity of FBP by affinity CE. Heparin is a highly sulfated glycosaminoglycan and thus prone to induce anodic migration shifts of complexing analytes. Prior complexation of FBP with folate abolished heparin binding, and thus folate competes with heparin for binding to FBP. It was estimated that heparin bound several orders of magnitude less strongly than folate with an average dissociation constant in the 1-10 microM range. In contrast to the mobility shifts induced by heparin, free and folate-bound FBP were not separated by CE. However, binding of folate induced a distinct increase in FBP-peak symmetry, and using heparin as an affinity displacer, the free FBP in equilibrium with folate-FBP complexes could readily be separated from the complexes. While the folate-FBP interaction was too strong to be characterized quantitatively because of inadequate detection limits of a UV-based detection system, it was possible to estimate the folate-FBP binding stoichiometry using this approach. The heparin interaction fractionated FBP into distinct subfractions, and the CE approach thus promises to be useful for unraveling the complex oligomerization behavior of FBP isoforms as well as for evaluating the FBP affinity for various species and analogs of glycosaminoglycans and folate.

Contribution of Organic Acid Anions to the Alkalinity of Natural Humic Water

It was found that the correction for organic acid anions to the alkalinity of water depends on the concentration of these anions, their dissociation constant, and pH; for humic water, it is expressed by the equation [Δ HCO3−] = [Aorg−](0.094 – 9064 × 10−pH). Based on this equation and data on the alkalinity and ion balance of water, the concentrations of hydrogen carbonates and organic acid anions can be calculated. In accordance with the found average dissociation constant of humic acids (pK = 4.0), the contribution of their anions to the alkalinity of water is no higher than 10% of their concentration.

NMR Spectroscopic Study of Noble Gas Binding into the Engineered Cavity of HPr(I14A) from Staphylococcus carnosus

Xenon binding into preexisting cavities in proteins is a well-known phenomenon. Here we investigate the interaction of helium, neon, and argon with hydrophobic cavities in proteins by NMR spectroscopy. 1H and 15N chemical shifts of the I14A mutant of the histidine-containing phosphocarrier protein (HPr(I14A)) from Staphylococcus carnosus are analyzed by chemical shift mapping. Total noble gas induced chemical shifts, Delta, are calculated and compared with the corresponding values obtained using xenon as a probe atom. This comparison reveals that the same cavity is detected with both argon and xenon. Measurements using the smaller noble gases helium and neon as probe atoms do not result in comparable effects. The dependence of amide proton and nitrogen chemical shifts on the argon concentration is investigated in the range from 10 mM up to 158 mM. The average dissociation constant for argon binding into the engineered cavity is determined to be about 90 mM.

Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen

In semen, the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 mol (mol of protein)(-1) and with an average dissociation constant of approx. 5 microM per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.

The Role of Functional Monomer–Template Complexation on the Performance of Atrazine Molecularly Imprinted Polymers

The extent of functional monomer–template complexation in noncovalent molecularly imprinted polymer (MIP) prepolymerization mixtures have been examined using 1H‐NMR spectroscopy, and observed complex stabilities have been correlated to polymer–template recognition. The triazine herbicide atrazine was used as a template in molecular imprinting studies using the functional monomers methacrylic acid (MAA), 4‐vinyl pyridine (4‐VP), and an equimolar mixture of the two. Nuclear magnetic resonance (NMR) titration studies using acetic acid, an analogue for MAA, yielded an average apparent dissociation constant (app K diss) of 65 ± 7 mM for atrazine complexation in chloroform (293 K). Studies using pyridine, an analogue for 4‐VP, demonstrated no significant complex formation over the concentration range studied. Atrazine MIPs, and corresponding nonimprinted reference polymers, were synthesized using MAA, 4‐VP, or an equimolar mixture of the two, and were evaluated using high‐performance liquid chromatography (HPLC) and radio ligand binding studies. Importantly, the recognition characteristics of the polymers reflected the observed complex stabilities in the prepolymerization mixtures; thus, MAA MIPs displayed superior recognition and binding capacity compared to polymers prepared with 4‐VP. MIPs synthesized using a combination of MAA and 4‐VP demonstrated intermediate recognition. These results suggest that NMR spectroscopy may be used as a predictive tool for the screening of functional monomers for use in molecular imprinting protocols. Financial support [Swedish Science Council (VR), Carl Trygger's foundation, KK‐foundation, the Graninge Foundation, and the University of Kalmar] is most gratefully acknowledged. We also thank Anna Lilja for excellent technical assistance.

In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis.

Aptamers with high affinity for IgE were selected using capillary electrophoresis to demonstrate the compatibility of this technique with SELEX. The high selectivity and efficiency of CE gave rise to a very high rate of enrichment, allowing high-affinity, high-selectivity aptamers to be obtained in only four rounds of selection. Decreasing the number of rounds shortens the selection procedure from the 4-6 weeks typical of SELEX to several days. The use of "bulk" dissociation constant measurements was introduced as a method for assessing the DNA pool after each round of selection. The average dissociation constant of the sequences in the DNA pool for IgE after four rounds of selection was 29 nM. The distribution of the dissociation constants for the sequences in the pool was very narrow with a standard deviation of only 6 nM. All of the sequences assessed exhibited high specificity for human IgE when compared with human IgG or mouse IgE.

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10 6 M −1 s −1 and 6.7 ± 2.5 × 10 −2 s −1, respectively, which corresponded to an average equilibrium dissociation constant ( K D) of 2.6 ± 1.4 × 10 −8 M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

Properties of the metal ion binding sites of Zn-transcription factor IIIA (TFIIIA) were investigated to understand the potential of this type of zinc finger to undergo reactions that remove Zn2+ from the protein. Zn–TFIIIA was purified from E. coli containing the cloned sequence for Xenopus laevis oocyte TFIIIA and its stoichiometry of bound Zn2+ was shown to depend on the details of the isolation process. The average dissociation constant of Zn2+ in Zn-TFIIIIA was 10−7. The dissociation constant for Zn-F3, the third finger from the N-terminus of TFIIIA, was 1.0×10−8. The reactivity of Zn–TFIIIA with a series of metal binding ligands, including 2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene (zincon), 4-(2-pyridylazo)-resorcinol (PAR), and 3-ethoxy-2-oxo-butyraldehyde-bis-(N4-dimethylthiosemicarbazone) (H2KTSM2) revealed similar kinetics. The reactivity of PAR with Zn–TFIIIA declined substantially when the protein was bound to the internal control region (ICR) of the 5S ribosomal DNA. Both Cd2+ and Pb2+ disrupt TFIIIA binding to its cognate DNA sequence. The Pb2+ dissociation constant of Pb-F3 was measured as 2.5×10−8. According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb2+.