Determination of total and unbound calcium in nanoliter samples of cartilage fluid and serum

A methodology is described for accurate determination of total and unbound calcium concentration in volumes of about 20 to 30 nl of physiological fluids. The technique involves the use of a helium glow photometer, an original dialysis nanocell capable of processing volumes at the nanoliter level, and La³⁺ to displace calcium from its bound states. An equilibrium dialysis criterion is used to define the unbound calcium concept. Total and unbound calcium was determined in cartilage fluid samples aspiratedin vivo from the growth cartilage of three different rat preparations as well as in serum obtained from the correspondent animals. Calcium values reported here could be useful to the understanding of the biological processes of endochondral calcification, and the methodology developed could find wider application in analytical biology.     

Determination of protein-ligand binding constants at equilibrium in biological samples.

Protein-ligand complexes can be separated functionally into two classes. "Specific" binding is characterized, in relative terms, by a high affinity for the ligand and a low binding capacity. "Non-specific" binding is characterized by a low affinity and a very large capacity. The calculation of equilibrium binding constants for any specific protein-ligand interaction requires the exact determination of the unbound ligand concentration and the specifically bound ligand concentration. These determinations usually require corrections for the contribution of non-specific binding. The use of two correction terms, kn and f, is proposed: kn is the product of the affinity constant k times the number of binding sites n of the non-specific components, while f is the fraction of the non-specific binding included in the experimental estimates of bound ligand. Several theoretical solutions using these terms are proposed for the calculation of specific binding constants. The practical choice of the correction factor may be different when the simultaneous measurement of the affinity constant and maximum number of binding sites, or when only the latter, is desired. In the case of complex binding systesm containing more than one specific component, the individual constants can be determined by non-graphical methods, using computer-aided iterative statistical calculations. A complete solution is given for a system containing two specific plus non-specific interactions and actual experiments are reported for steroid hormone-receptro complexes.     

Filter assay method for the estrogen receptor protein: Detection of both filled and unfilled estrogen binding sites

New filter assay methods are presented for quantitating both cytoplasmic and nuclear forms of the estrogen receptor protein. These methods exploit the strong adsorption of this protein to glass-fiber filters, which appears to occur without loss of steroid binding affinity. A “direct assay protocol” is described that detects only unfilled (nonliganded) estrogen binding sites. In addition, a convenient “exchange assay protocol” has been developed that detects, in addition, those receptors present whose binding sites have already bound nonradioactive estradiol. For the exchange assay, an extract containing receptor is adsorbed to a filter, which is washed free of unbound steroid and then equilibrated for a prolonged period with an excess volume of buffer containing radioactive estradiol. After brief washing in steroid-free buffer, the radioactivity adsorbed to the filter is measured to determine the amount of receptor present. These assays can be used at either 4 or 23°C, over a broad range of salt concentrations. The background of nonspecific binding is extremely low, due in part to the almost negligible affinity of free estradiol for the glass-fiber support.     

Cicletanine binding to human plasma proteins and erythrocytes, a particular HSA-drug interaction

The binding of cicletanine to human serum, isolated proteins and red blood cells was studied in vitro by equilibrium dialysis. Our results show this drug is highly bound to serum (97.3%) at therapeutic levels. No saturation to the binding sites was seen. Human serum albumin was shown to mainly responsible for this binding (93.5%) with a saturable process characterized by one binding site with a moderate affinity (K = 75800 M-1) and a non saturable process with a low total affinity (nK = 6400 M-1). Like many basic lipophilic drugs, cicletanine showed a saturable binding to alpha-1-acid glycoprotein with one site and a moderate affinity (K = 38,800 M-1). Its binding to lipoproteins and red blood cells was weak and non saturable. Over the range of therapeutic concentrations, the unbound fraction in blood remains constant (3.6%). Moreover, interactions were studied using bilirubin and non esterified fatty acids at pathological concentrations and these endogenous compounds did not alter cicletanine binding human serum or to human serum albumin likewise cicletanine shared the diazepam-site on HSA but no inhibition could take place between cicletanine and the drugs sharing the same binding site in serum at therapeutic levels.     

Some properties of a glycoprotein with calcium binding ability found in human biological fluids in macroglobulinaemia IgM

An acidic glycoprotein with calcium-binding properties was isolated from the urine of patients with severe macroglobulinaemia IgM. The molecular weight of this protein determined by Sephadex gel filtration was found to be 62 000 ± 2800 in Tris · HCl buffer and 21 000 ± 1 000 in 6 M guanidine · HCl. The amino acid and carbohydrate composition of the isolated glycoprotein is presented. Electrophoretic migration of this protein was observed to be greatly affected by calcium ions present in the buffer in a concentration of 10^?3 M. At least two sets of binding sites seem to participate in binding calcium. The values 2.2 · 10⁶ M^?1 for the apparent association constant and 4.4 · 10^?4 mol of Ca²⁺ bound per g of protein for high affinity binding sites were estimated, on the basis of data from the equilibrium dialysis. The origin possible biological role of this protein is discussed.     

A dialysis cell for nuclear magnetic resonance spectroscopic measurement of protein-small molecule binding

A dialysis cell is described for use in an NMR spectrometer, to make spectroscopic determinations of protein-small molecule binding. The protein solution is contained within a cylindrical dialysis tube which is concentrically suspended in an NMR tube containing a protein-free dialysis buffer. Simultaneous determinations of the equilibrium transmembrane distribution of the small molecule and the chemical shifts in both compartments are made spectroscopically, providing estimates of the dissociation constant and the chemical shift of the bound species. The cell is used for 31P NMR spectroscopic measurement of the degree of binding of 2,3-diphosphoglycerate to hemoglobin in a 2.8 mM carboxyhemoglobin solution at pH 6.9 and 21 degrees C. The Kd is found to be 2.4 x 10(-3) M.     

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid.     

The measurement of free nitrendipine in human serum by an equilibrium dialysis - radioreceptor assay

A radioreceptor assay using [³H]nitrendipine and rat cerebral cortical membranes, in conjunction with equilibrium dialysis, measures the unbound (free) level of nitrendipine in human sera. The sensitivity of the assay is 0.1–0.2 picomoles/ml and is linear from 4 × 10^?11 to 4 × 10^?9 M nitrendipine. Other dihydropyridine calcium channel antagonists may be measured using this assay if these compounds are used to generate the standard curve. Blank serum interferes with specific [³H]nitrendipine binding (24 percent inhibition per 20 μ1 serum) whereas serum dialysates do not. Total serum nitrendipine levels may be measured, but the sensitivity of the assay is decreased due to interference by serum. Nitrendipine is highly protein bound in serum (93 – 99 percent). This protein binding is essentially unchanged over a serum concentration from 1 to 100 ng/ml. This assay is suitable for pharmacokinetic and pharmacodynamic studies.     

Some properties of a glycoprotein with calcium binding ability found in human biological fluids in macroglobulinaemia IgM.

An acidic glycoprotein with calcium-binding properties was isolated from the urine of patients with severe macroglobulinaemia IgM. The molecular weight of this protein determined by Sephadex gel filtration was found to be 62 000 +/- 2800 in Tris - HCl buffer and 21 000 +/- 1000 in 6 M guanidine - HCl. The amino acid and carbohydrate composition of the isolated glycoprotein is presented. Electrophoretic migration of this protein was observed to be greatly affected by calcium ions present in the buffer in a concentration of 10(-3) M. At least two sets of binding sites seem to participate in binding calcium. The values 2.2 - 10(6) M-1 for the apparent association constant and 4.4 - 10(-4) mol of Ca2+ bound per g of protein for high affinity bindings sites were estimated, on the basis of data from the equilibrium dialysis. The origin and possible biological role of this protein is discussed.     

Equilenin: a specific fluorescent probe for steroid—protein interactions in sex steroid-binding protein

Equilenin, a naturally fluorescent steroid, has high binding affinity for human sex steroid-binding protein (SBP). At 4°C the equilibrium association constant is ～6 × 10⁷ M^?1. The fluorescence excitation and emission spectra of the steroid—protein complex indicate that both hydrophobic interactions and hydrogen bonding of the 3'-hydroxyl group of the estrogen are important in its binding to the protein. Equilenin has a substantially different 3-dimensional spatial configuration compared with the normally bound androgens, and yet exhibits very tight binding to SBP. This suggests that SBP undergoes a conformational change to accomodate equilenin.     

Test of a theory relating to the cross-linking of IgE antibody on the surface of human basophils

Recent mathematical models of bivalent hapten-induced histamine release from basophils predict that under appropriate conditions histamine release is maximum when cross-link formation is maximum, at a hapten concentration equal to 1/(2Ka), where Ka is the average affinity constant of the hapten for a single IgE binding site. To test this prediction we sensitized human basophils with a monoclonal anti-dinitrophenol IgE and generated histamine release dose-response curves with a bivalent hapten, alpha, epsilon-DNP-lysine. The monoclonal IgE has a published affinity constant of 7.1 X 10(7) M-1 for epsilon-DNP-lysine as determined by equilibrium dialysis. From the position of the maximum of the histamine dose-response curves, both in the presence and in the absence of monovalent DNP hapten, we determine that the sensitizing IgE has an intrinsic affinity constant of 6.9 +/- 0.5 X 10(7) M-1 for epsilon-DNP-lysine and 1.2 +/- 0.6 X 10(6) M-1 for alpha-DNP-lysine. The agreement between the two estimates of the epsilon-DNP-lysine affinity constant, one from histamine release experiments involving surface bound IgE and one from binding experiments involving IgE free in solution, 1) is consistent with a central prediction of the theory of cross-linking and 2) indicates that the hapten-binding properties of the IgE are unaffected by its being bound to Fc epsilon receptors on the basophil surface.     

Plasma protein binding interaction of prednisone and prednisolone

The plasma protein binding interaction of prednisone and prednisolone were characterized by equilibrium dialysis. Prednisone and prednisolone are bound equally but weakly to human albumin (affinity constant, K approximately equal to 1 X 10(3) M-1). Transcortin affinity for prednisolone is 10-fold greater (51.3 X 10(6) M-1) than that for prednisone (4.3 X 10(6) M-1). In competition under pharmacologic conditions, prednisolone inhibits prednisone binding to transcortin producing linear binding averaging 55%. Prednisone does not affect prednisolone binding and does not complicate pharmacokinetic studies of the latter.     

Evaluation of quantitative affinity chromatography by comparison with kinetic and equilibrium dialysis methods for the analysis of nucleotide binding to staphylococcal nuclease.

The elution of staphylococcal nuclease on thymidine 3'-(p-Sepharose-aminophenyl phosphate) 5'-phosphate (nucleotide ligand of nuclease covalently bound to Sepharose 4B) was studied in the presence of a variety of soluble nucleotide ligands. The elution volumes of nuclease vary proportionally with matrix-bound ligand concentration (at constant soluble ligand concentration), inversely with soluble ligand concentration (at constant matrix-bound ligand concentration), and inversely with dissociation constant of soluble ligand (at constant concentrations of soluble and matrix-bound ligand). The variation of elution volume was related to an expression which described the competition of soluble and matrix-bound ligand for nuclease binding. Using this expression, values for dissociation constants were derived for nucleotide ligands in both the soluble and bound form. The values for soluble ligand were found to correspond closely to those obtained by either equilibrium dialysis or kinetics of inhibition of nuclease activity. Furthermore, a close correspondence was found between the values of dissocation constants for matrix-bound and soluble thymidine 3'-(p-aminophenyl phosphate) 5'-phosphate, thus defining the interaction of nuclease with the matrix-bound ligand as a process quite similar to that occurring in solution.     

Effect of active immunisation against testosterone-3-BSA on circulating levels of testosterone, LH, prolactin and testosterone antibody titre in the male rat

Male Sprague-Dawley rats were actively immunised against testosterone-3-bovine serum albumin (T-3-BSA) and on appearance of detectable anti-testosterone antibodies, elevated serum testosterone and LH concentrations were observed. These concentrations reached values of >28 μg/100ml testosterone and 16 μg/100ml LH in some animals after 5 months of immunisation. The corresponding prolactin values did not appear to differ significantly from controls. The circulating bound testosterone fraction as determined by equilibrium dialysis, rose from 65.0 ± 2.75% before immunisation to 98.7 ± 0.75% in those animals possessing high titre antisera. This entailed a nett $\text{decrease}$ in the concentration of unbound steroid from 144 ± 49 ng/100 ml to 78 ± 25 ng/100ml.     

The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein.

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.     

Ligand-affinity sedimentation equilibrium using an air-driven ultracentrifuge

An air-driven ultracentrifuge has been used to study the distribution of radioactive ligands at sedimentation equilibrium. In the presence of a suitable acceptor and under conditions where the ligand is essentially all bound the distribution of ligand can be analyzed to yield the molecular weight of the acceptor molecule. Suitable conditions can be chosen either experimentally by measuring the ratio of bound ligand compared to unbound ligand or theoretically for systems in which the ligand-binding affinity and number of acceptor binding sites is known. The method is applicable to the molecular characterization of binding proteins in crude mixtures and results are presented for the binding of various fatty acids to serum albumin samples.     

Cadmium binding to human alpha 2-macroglobulin

alpha 2-Macroglobulin (alpha 2M) is one of the major cadmium-binding proteins of human plasma. As determined with equilibrium dialysis, alpha 2M bound 4.6 (+/- 0.7) mol Cd2+ per mol protein with an apparent dissociation constant of (9.6 (+/- 5.0] X 10(-7) M. Methylamine-modified alpha 2M (alpha 2M-Me) had a similar affinity for Cd2+ (Kd,app = 5.3 X 10(-7) M), but fewer binding sites. Cadmium produced a small increase in the amidolytic activity of trypsin in the presence of alpha 2M and soybean trypsin inhibitor. Using the binding parameters determined from the equilibrium dialysis studies, the Cd2+ concentration which produced a half-maximal increase in amidolytic activity corresponded to saturation of all Cd2+-binding sites in one-half of the alpha 2M molecules. From these results, a model is proposed in which one Cd2+-binding site is present in each of the four polypeptide chains which compose alpha 2M.     

Study of factors affecting binding of zinc with albumin at physiological zinc concentrations

Albumin has very high affinity for many organic and inorganic compounds that may influence albumin bound Zn (ABZn). To get insight of these molecular interactions, the effect of riboflavin, nicotinic acid, thiamine, folic acid, pyruvic acid and glucose on ABZn were studied. The ABZn was separated from the unbound zinc using equilibrium dialysis and estimated using atomic absorption spectrometer. At therapeutic zinc concentrations, folic acid and thiamine significantly enhanced the ABZn (p < 0.010), while nicotinic acid inhibited zinc binding to albumin. Folic acid was found to enhance the ABZn also at lower zinc concentrations representing physiological levels of plasma zinc (138-150 micromoles) (p < 0.05).     

Ligand binding by estrogen receptor beta attached to nanospheres measured by fluorescence correlation spectroscopy.

Although many indirect methods have been chosen to study the system of estrogen receptor ligand binding, an ideal method is fluorescence correlation spectroscopy (FCS). FCS is nondestructive to the sample, uses very small sample volumes, and operates well within physiological concentration ranges. The methodology was developed to biotinylate the estrogen receptor beta-ligand binding domain (ERbeta-LBD) using biotin with a very short spacer and to then attach this protein to a 40 nm neutravidin-coated bead (nanosphere). Diffusional FCS data were obtained for a fluorescently labeled coactivator peptide, steroid receptor coactivator peptide-1 (A-SRC-1(2)), in the absence and presence of bead-bound ERbeta-LBD. Data were also acquired in the presence of one of the endogenous ligands for ERbeta, 17beta-estradiol, and with tamoxifen. The bead strategy resulted in a decreased receptor diffusion coefficient and consequent increase in the decay time of the FCS autocorrelation functions for receptor-bound, labeled SRC-1(2). Thus, free and bound coactivators were much more readily distinguished by FCS. Discrimination between the fluorescently labeled unbound and bound species could be determined in autocorrelation functions obtained in as few as 30 s. The advantage of using FCS with the ERbeta-LBD: bead methodology is the ability to obtain reliable and reproducible data in a short time frame.