Numerical and experimental demonstrations of the need for caution in the use of zonal gel chromatography for characterizing ligand interactions with small acceptors.

Quantitative expressions are presented for the evaluation of equilibrium constants for interactions of the type A + B in equilibrium C from experiments entailing the application of a small zone of acceptor-ligand mixture to a column of gel preequilibrated with ligand solution [J.P. Hummel and W.J. Dreyer (1962) Biochim. Biophys. Acta 63, 530-532]. Only in the event that identical elution volumes pertain to acceptor and complex does the steady-state binding constant (Kss) obtained by that method equal the thermodynamic equilibrium constant (K). Simulated elution profiles are then generated with parameters relevant to gel chromatography of the ATP-Mg2+ system on Sephadex G-10 in order to demonstrate the practical importance of the need for distinction between Kss and K in situations where acceptor and complex do not comigrate. A study of the interaction between soybean trypsin inhibitor and cytochrome c by gel chromatography on Sephadex G-75 is then used to illustrate the feasibility of combining information from Hummel-Dreyer experiments with the theoretical expressions to characterize systems under the more general conditions that the elution volumes of A and C differ. A finding of considerable theoretical interest in relation to the simulation of mass migration behavior is the demonstration that a truncation error is the source of zonal spreading in the theoretical-plate model of chromatography. This truncation error is shown to be the source of spreading generated whenever solution of an abbreviated (diffusion-free) continuity equation involves substituting first differences for first derivatives in the differential equation describing mass transport.

Protein immobilization on silica supports. A ligand density study.

The immobilization of proteins on diol-bonded silica matrices containing carboxyl groups (spacer arms) was studied. It was found that the activated ester coupling method worked best with N,N'-dicyclohexylcarbodiimide as the condensing agent in the activation step. During protein coupling, the amount of protein immobilized was highest below pH 6. The optimum pore size of the silica was 300-1000 A. The spacer arm ligand density was varied over as much as a 100-fold range and the effects on the total activities and specific activities of several proteins were studied. Two proteins exhibited up to two-fold increases in specific activity at low ligand densities. However, the total amounts of activity and protein immobilized decreased at low ligand densities.

Distribution of [3H]dihydrotetrabenazine binding in bovine striatal subsynaptic fractions: enrichment of higher affinity binding in a synaptic vesicle fraction.

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3-9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95-400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.

Bioimmunoassay (BIA): a sandwich immunoassay scheme employing monoclonal antibodies and hormone receptors to quantify analytes.

When some antigens bind to receptors, a portion of the antigen remains exposed and can be recognized by labeled monoclonal antibodies. By measuring the amount of antibody bound to the antigen-receptor complex, one can quantify the amount of antigen that is present. Since this assay procedure depends on simultaneous receptor recognition of a biologically active site and antibody recognition of a distal epitope on the analyte, we call it a bioimmunoassay. Bioimmunoassays have many of the advantages of radioligand receptor assays (RRA) used to quantify biological activity and, depending on the choice of antibodies employed, may be more specific than RRA. In addition, since they are sandwich assays, they are usually more sensitive than RRA. Bioimmunoassays can be performed in several different modes and in the case described here we used a radiolabeled antibody to detect hormone-receptor complexes. Hence we term this example a bio-immunoradiometric assay or BIO-IRMA. We illustrate the properties of various assay procedures using a monoclonal antibody to the beta subunit of hCG which recognizes an epitope common to all other mammalian LH/hCG-like gonadotropins and which is capable of detecting 10 pg of hCG standard. In principle, this assay can be applied to any material capable of binding to a receptor, enzyme, etc. which can also be recognized by an antibody. Since it is a sandwich type of assay, it is subject to the same advantages and limitations of other sandwich assays except that it can be used to discriminate some biologically active and inactive analytes. Monoclonal antibodies which are prepared from spleen cells of animals immunized with antigen-receptor complexes and selected for their ability to bind antigen-receptor complexes should prove most useful for bioimmunoassay procedures.

TCDD receptor ligands present in extracts of urban air particulate matter induce aryl hydrocarbon hydroxylase activity and cytochrome P-450c gene expression in rat hepatoma cells.

In previous studies we have shown that substances associated with particulates collected from urban air and automobile exhaust bind with high affinity to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor present in rat liver cytosol. In this study we used a rat hepatoma cell line, H4IIE, to investigate the effect of such substances on an enzyme system, aryl hydrocarbon hydroxylase (AHH), which is regulated via the TCDD receptor. The results demonstrate that AHH activity in the H4IIE cell line can be induced by extracts of particulates collected from urban air and automobile exhausts in a dose-dependent manner, and that the AHH activity is inducible by five polycyclic aromatic hydrocarbons (PAHs) including 1-/3-nitrobenzo[a]pyrene and 6-chlorochrysene, all present in extracts of particulates from urban air and automobile exhausts. The induction of AHH activity is correlated to apparent TCDD receptor affinity for investigated PAHs (r = 0.85) and particulate extracts. Biochemically, treatment of the cells with 5,6-benzoflavone significantly increased the level of cytochrome P-450c but not P-450d as shown by immunoblotting and analysis of mRNA levels. The data indicate that substances present in extracts of urban air particulates can interact with the TCDD receptor in intact cells and cause an accumulation of cytochrome P-450c mRNA leading to an increased synthesis of the gene product and thus an increase in enzyme activity.

Pitfalls in demonstrating an endogenous ligand of imipramine recognition sites.

The recognition sites for the 5-hydroxytryptamine (5-HT) uptake inhibitors imipramine and paroxetine may represent receptors for a presently unknown endogenous ligand, whose function would be to modulate 5-HT uptake. Attempts to isolate such a factor from rat brain tissue are described, following a published procedure. It is shown that chromatographic fractions found to inhibit the binding of [3H]imipramine and [3H]paroxetine to rat brain membranes consisted of material essentially unretained by the reverse-phase HPLC column, and they were of high osmolarity. Thus, the activity was probably unspecific in nature, and the presence in rat brain of the factor has not been unequivocally demonstrated.

Protocols for use of ultrafiltration in determination of free ligand concentration and of complexity of ligand/protein interactions.

Analysis of a simple ligand/binding-protein association model predicts a decrease in the percentage of free hormone during ultrafiltration. Re-analysis of the original data validating ultrafiltration in free-ligand determinations (Sophianopoulos et al., Arch Biochem Biophys 1978;187:132-7) confirms this prediction. Binding data indicating a constant percentage of free hormone (Hammond et al., J Biol Chem 1980;255;5023-6) require a more complex ligand-protein interaction model. Application of these procedures to any new ligand system requires measurement of the percentage free, or of free-ligand concentration, as a function of filtration time or volume. The protocol becomes important if values for percentage free are to be accurate.

Analysis of competition binding assays: assessment of the range of validity of a commonly invoked assumption.

A common assumption invoked in the analysis of competition binding assays is that the fractional saturation of sites with the unlabeled ligand is given by 1-(the concentration of bound labeled ligand in the presence of unlabeled ligand)/(the concentration of bound labeled ligand in the absence of unlabeled ligand). This assumption is critically evaluated in the context of several binding models: (a) binding of univalent ligands to multiple classes of equivalent and independent sites, with and without nonspecific binding; (b) cooperative binding of univalent ligands; and (c) binding of multivalent ligands to a single class of univalent acceptors. We show that the conventional assumption is only valid when the labeled ligand is mainly in the free form, occupies a small fraction of the total sites and binds univalently to all sites in an equivalent and independent manner, and when the unlabeled ligand forms 1:1 complexes with the acceptor sites. When these conditions are satisfied, the conventional assumption is valid even if the unlabeled ligand binds to nonequivalent sites or exhibits cooperativity. Finally, we apply the theory derived for case (a) above to the binding of fluoresceinated epidermal growth factor to A431 cells and demonstrate that the analysis of data obtained from both conventional and competition assays provides information which is difficult, if not impossible, to obtain from either assay alone.

Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins.

Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin.

Affinity gel titration: quantitative analysis of the binding equilibrium between immobilized protein and free ligand by a continuous titration procedure.

A new method, called affinity gel titration, for analyzing the specific interaction between an immobilized protein and a ligand molecule is presented. Only one or two experimental runs permit the determination of not only the equilibrium constant but also the amount of immobilized protein. A suspension of the immobilized protein on agarose gel beads is confined in a constant volume mixing cell. A solution of a specific ligand molecule of constant concentration is introduced into the cell so that its concentration in the cell increases continuously (as in a mixing chamber for forming a convex gradient). The correlation between the concentration of the ligand in the efflux and the cumulative volume of the efflux can be analyzed either by regression to a theoretical curve or by a graphical method. Specific binding of p-aminobenzamidine to immobilized Streptomyces griseus trypsin was studied by this method. The dissociation constant and the amount of active trypsin were determined. The values obtained were in good agreement with the inhibition constant obtained by a kinetic experiment with free trypsin and with the amount of active site measured by using p-nitrophenyl p'-guanidinobenzoate, respectively. A single run of the titration procedure could be completed within 1 h.

An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins.

A new method for the measurement of rapid isotopic release from a membrane compartment is described. Membrane vesicles loaded with isotope, or broken membranes with bound radioactive ligand, are filtered onto the surface of a cellulose ester filter; the rate of the loss of isotope from the membrane compartment is followed continuously by collecting fluid which is passed through the filters under high pressure. A change in release rate is initiated by changing the solution or by delivering a flash of light to a photosensitive sample. The approach has been used to study rapid 22Na efflux from membrane vesicles rich in the ouabain-sensitive Na pump, and to examine dissociation of 32P and 86Rb from membrane-bound Na,K-ATPase. Since the rate of efflux is measured, and not the total counts remaining on the filter, the technique has high sensitivity. A complete time course is obtained using only a few micrograms of membrane protein. The apparatus described is simple, inexpensive, and easily constructed; with the present device, time resolution is approximately 10 ms.

Identification of copper- and zinc-binding ligands in human and bovine milk.

The object of this study was to identify copper and zinc ligands detected during modified gel chromatography ( MGC ) of bovine and human milk ultrafiltrates. Isolation by anion-exchange chromatography and subsequent proton nuclear magnetic resonance (NMR) spectroscopy analysis demonstrated that the sole major low-molecular-weight ligand binding copper and zinc in bovine milk is citrate. Human milk apparently also contains citrate as a major metal-binding ligand but also contains amino acids, of which primarily glutamate was purified by anion-exchange chromatography. The amino acids bind copper well but are weak zinc-binding ligands. An artifactual MGC peak was seen in the milks, which was shown to be caused by the calcium present in the milk samples. No major differences in zinc-binding capacity were demonstrated between the low-molecular-weight fractions of the two milks. Although citrate may play a role in zinc uptake, it is apparently not the difference between the milks crucial to the acrodermatitis enteropathica individual. The difference in zinc availability between the milks may lie in some other aspect such as binding by proteins, which were noted to bind metal during MGC of nonultrafiltered milks.

Thin-layer fluorescence cell for ligand binding studies.

A high precision method for measuring the binding of gaseous ligands to proteins is presented. Front face fluorescence techniques are utilized with a special thin-layer cell in order to monitor the change in fluorescence intensity caused by changing the ligand partial pressure. The method is illustrated by examining the binding of carbon monoxide to hemocyanin from the lobster Homarus americanus.

Source of analytic variation in ligand assay: a study based on data from the 1976 and 1981 Basic Ligand Assay Surveys of the College of American Pathologists.

Six paired specimens distributed to laboratories in 1981 at approximately three-month intervals and four paired specimens distributed to laboratories in 1976 at intervals of three to nine months for analysis by the College of American Pathologists Survey form the basis for this study. Twelve of 37 (35%) of pool-analyte-technic combinations yielded significantly changed mean values in 1976, while 34 of 196 (17%) pool-analyte-technic combinations yielded significantly changed values in 1981. Probable instability in thyroxine and folate was demonstrated in the 1981 control pools. Precision generally continued to improve from 1976 to 1981. The poorest precision now is observed in the analysis of peptide hormones. A majority of the observed analytic variation during 1981 for most analytes relates to extralaboratory factors. Improvement in performance is largely dependent on intermanufacturer standardization of procedures and long-term maintenance of equivalence of the results of kit procedures by each manufacturer.

Correction for light absorption in fluorescence studies of protein-ligand interactions.

It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.

[How far has the search for endogenous ligands of benzodiazepine receptors progressed?]. / Où en est la recherche des ligands endogènes des récepteurs des benzodiazépines?

The discovery, in 1977, of the specific binding sites for benzodiazepines in the brain of mammals, notably in man, lends support to the possible existence of endogenous compounds acting as natural ligands of these sites. At present, a dozen of molecules having the capacity to displace bound 3H-benzodiazepines from their specific sites have been extracted from the brain of several species (rat, pig, bovine...), the cerebrospinal fluid and urine of man. These molecules are proteins, peptides, purines, beta-carbolines... and exhibit (some) pharmacological properties similar or opposite to those of benzodiazepines. The most recent data concerning benzodiazepine receptors suggest that the endogenous ligand would be, if it exists, a benzodiazepine-like compound (agonist) with an indolic structure.

Calcium fractions in seminal plasma and functional properties of human spermatozoa.

The concentration of ionized calcium [Ca++] was reproducibly determined in human seminal plasma with a calcium sensitive electrode (Orion Space Stat 20). Dilution of the seminal plasma gave a linear decrease in free calcium concentration. Freezing and thawning or storage of the seminal plasma under anaerobic conditions did not influence the level of ionized calcium. Storage under aerobic conditions gave a temperature dependent decrease in CA++ which parallelled a spontaneous increase in pH. The mean concentration of ionized calcium (measured after a standardized air exposition) was 0.17 mM +/- 0.05 (SD) (range 0.09-0.29 mM). The Ca++ level was not correlated to the total calcium concentration or to markers for prostatic (zinc) or seminal vesicular (fructose) secretions. There were, however, more motile spermatozoa in semen samples with an ionized calcium level less than the average (0.17 mM) than in semen samples with a higher Ca++ level (53.8% +/- 8.4, N = 17 vs 45.0% +/- 12.8, N = 15, respectively, p less than 0.05). The percentage live spermatozoa was also higher (62.4% +/- 10.4, N = 17 vs 50.7% +/- 14.2, N = 15, p less than 0.01) in the semen samples with a low [Ca++]. Spermatozoa in the low [Ca++] group did also exhibit a better progressive motility than spermatozoa in the high [Ca++] group (p less than 0.05). It is suggested that the low level of ionized calcium in seminal plasma is of importance for motility of human spermatozoa and that the transfere of spermatozoa from a high Ca++ in the epididymis to the low levels in accessory sex glands secretion might be of significans for activation of spermatozoa upon ejaculation.