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PubMed1996

An enzyme immunoassay based micro-neutralization test for titration of antibodies to human cytomegalovirus (CMV) and its correlation with direct ELISA measuring CMV IgG antibodies.

An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, ph 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations.

Antibodies, Viral↗
PubMed1999

A quantitative focus assay for titration of retroviruses that encode human granulocyte-macrophage colony-stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a haematopoietic growth factor that regulates proliferation, differentiation, and effector functions of monocyte-macrophages and granulocytic cells. Because of the ability of this cytokine to enhance immune functions of antigen-presenting cells, retroviruses encoding GM-CSF have been used to transduce GM-CSF into murine and human tumour cells as part of autologous tumour vaccine strategies. We have previously shown that NIH 3T3 cells engineered to express functional human GM-CSF receptors (hGMR-NIH 3T3), become fully transformed when these cells are incubated in the presence but not in the absence of human GM-CSF. In this study we have used these hGM-CSF conditional transformants to devise a sensitive focus assay to titrate retroviruses encoding hGM-CSF, using MFG-hGM-CSF/Psi-CRIP as our model virus. This helper-free amphotropic retrovirus, which has been frequently used to transduce hGM-CSF into tumour cells, was quite transforming in our indicator cell line, exhibiting virus titres well above 10(5)FFU/ml. The transformation-based assay described here allows rapid determination of the titre of hGM-CSF-viruses, and may serve as a model for development of quantitative assays for other cytokine-encoding viruses of clinical importance.

3T3 Cells↗
PubMed1997

Investigation of Surface-Molecule Interactions on the Silica Gel Surface by the Gas Phase Titration Method

The distribution of adsorption sites on the silica gel surface was investigated by applying the chromatographic gas phase titration method. Ethylmethylketone was the blocking agent, and n-octane, benzene, and chloroform were the testing agents. These substances were chosen because of their various types of intermolecular interactions, i.e., nonspecific, electron-donor, and electron-acceptor, respectively. The retention data for all testing agents were directly compared, apart from the differences in volatility of these adsorbates. Three kinds of adsorption sites, differing in their interaction energy, were found on the silica gel surface for each testing agent. The results obtained were discussed in terms of various types of surface-molecule interactions. Then, the changes in free enthalpy of adsorption were calculated for all testing agents. Based on these data, the influence of ethylmethylketone on the interactions of the silica gel surface was estimated.

Journal Article↗
PubMed1998

Surface Titrations of Perlite Suspensions.

The surface charge behaviour of unexpanded and expanded perlite samples in KNO3 and NaCl solutions were investigated as a function of pH and ionic strength. The solutions of KNO3 and NaCl ranging from 10(-3) to 1.0 M were used. The potentiometric titration method was used to determine the surface charge of perlite samples. It was confirmed that the perlite samples had no the point of zero charge and was negatively charged in the pH range of 3-10. The double extrapolation method was used for determining the intrinsic equilibrium constants for simple ionization and complex ionization reactions. The values obtained are pKinta2 = 2.5 and p*KintK+ = 2.3 in KNO3 solutions and pKinta2 = 3.0 and p*KintNa+ = 2.4 in NaCl solutions for unexpanded perlite, and pKinta2 = 2.6 and p*KintK+ = 2.4 in KNO3 solutions and pKinta2 = 2.7 and pKintNa+ = 2.4 in NaCl solutions for expanded perlite. Copyright 1998 Academic Press.

Journal Article↗
PubMed1999

Titration Microcalorimetry Studies of the Interaction between Humicola lanuginosa Lipase and Ionic Surfactants.

Microcalorimetry has been used to study the interaction between Humicola lanuginosa lipase and either the anionic surfactant sodium dodecyl sulfate, SDS, or the cationic surfactant tetradecyltrimethylammonium bromide, TTAB. For SDS no conclusive evidence was obtained of whether or not an enzyme-surfactant complex is formed. Although not unambiguous, the calorimetric titration curves obtained with TTAB indicate formation of such a complex. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Thermodynamics of Interaction between Some Cellulose Ethers and SDS by Titration Microcalorimetry.

A titration calorimetric study of the interaction between nonionic cellulose ethers and ionic surfactant (SDS) has been extended to a larger number of polymers to explore the effect of variation of polymer hydrophobicity on the energetics of the process. "Hydrophobicity" as used here is an overall effect of the nature, degree, and number of substituents and is characterized by the cloud point and (aqueous) surface tension lowering abilities of the polymer. A direct correlation is found between the extent of "hydrophobicity" and the endo-enthalpic peak in the initial SDS concentration region of interaction. However, the overall mechanism of interaction is similar for all the polymers, being dominated by polymer-surfactant interactions initially and converting into a surfactant-surfactant interaction process at higher SDS concentrations. The importance of polymer characteristics thus becomes weaker at the later stages of the process. Differences between the polymers is also reduced by an increase of temperature, leading to a near overlap of observed enthalpy curves at 40 degrees C. The energetics of interaction are also mirrored by the isothermal surfactant binding curves and the changes in macroscopic and microviscosity of the system. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Thermodynamics of Interaction between Some Cellulose Ethers and SDS by Titration Microcalorimetry.

The interaction between certain nonionic cellulose ethers (ethyl hydroxyethyl cellulose and hydroxypropyl methyl cellulose) and sodium dodecyl sulphate (SDS) has been investigated using isothermal titration microcalorimetry at temperatures between 25-50 degrees C. The observed heat flow curves have been interpreted in terms of a plausible mechanism of the interaction of the substituent groups with SDS monomers and clusters. The data have been related to changes occuring in the system at the macro- and microscopic levels with the addition of surfactants and with temperature. The process consists predominantly of polymer-surfactant interactions initially and surfactant-surfactant interactions at the later stages. A phenomenological model of the cooperative interaction (adsorption) process has been derived, and earlier published equilibrium binding data have been used to recover binding constants and Gibbs energy changes for this process. The adsorption enthalpies and entropies have been recovered along with the heat capacity change. The enthalpic cost of confining the nonpolar regions of the polymers in surfactant clusters is high, but the entropy gain from release of hydration shell water molecules as well as increased freedom of movement of these nonpolar regions in the clusters gives the process a strong entropic driving force. The process is entropy-driven initially and converts to being both enthalpy and entropy-driven at high SDS concentrations. An enthalpy-entropy compensation behavior is seen. Strongly negative heat capacity changes have been obtained resulting from the transfer of nonpolar groups from aqueous into nonpolar environments, as well as a reduction of conformational domains that the chains can populate. Changes in these two components cause the heat capacity change to become less negative at the higher binding levels. The system can be classified as exhibiting nonclassical hydrophobic binding at the later stages of binding. Copyright 1999 Academic Press.

Journal Article↗
PubMed2000

A Titration Microcalorimetric Study of the Effects of Halide Counterions on Vesicle-Forming Aggregation in Aqueous Solution of Branched-Chain Alkylpyridinium Surfactants.

Titration microcalorimetry is used to study the influences of iodide, bromide, and chloride counterions on the aggregation of vesicle-forming 1-methyl-4-(2-pentylheptyl)pyridinium halide surfactants. Formation of vesicles by these surfactants was characterised using transmission electron microscopy. When the counterion is changed at 303 K through the series iodide, bromide, to chloride, the critical vesicular concentration (cvc) increases and the enthalpy of vesicle formation changes from exo- to endothermic. With increase in temperature to 333 K, vesicle formation becomes strongly exothermic. Increasing the temperature leads to a decrease in enthalpy and entropy of vesicle formation for all three surfactants. However the standard Gibbs energy for vesicle formation is, perhaps surprisingly, largely unaffected by an increase in temperature, as a consequence of a compensating change in both standard entropy and standard enthalpy of vesicle formation. Interestingly, standard isobaric heat capacities of vesicle formation are negative, large in magnitude but not strikingly dependent on the counterion. We conclude that the driving force for vesicle formation can be understood in terms of overlap of the thermally labile hydrophobic hydration shells of the alkyl chains. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Isothermal Titration Microcalorimetric Studies of the Effect of Temperature on Hydrophobic Interaction between Proteins and Hydrophobic Adsorbents.

This study attempted to comprehend how temperature affects hydrophobic interaction between proteins and hydrophobic adsorbents. By equilibrium batch analysis, we measured the adsorption isotherm to evaluate the protein-adsorbent affinity, while isothermal titration calorimetry was used to measure the adsorption enthalpy. In addition, the affinity and enthalpy differences between two proteins, alpha-chymotrypsinogen A and trypsinogen, with two adsorbents, butyl-Sepharose and octyl-Sepharose gel, under varying temperatures were studied with respect to the exposed hydrophobic segments of the protein and ligand hydrophobicity. The enthalpies obtained in this investigation can be used to more thoroughly understand the hydrophobic interaction between proteins and adsorbents. First, the adsorption isotherm experiments reveal that the adsorption quantity of the proteins with the Sepharose gels increases with temperature. For a microcalorimetric measurement, as temperature is increased from 298 to 310 K, the DeltaH value of alpha-chymotrypsinogen A with butyl-Sepharose increases, while the DeltaH value of trypsinogen is reduced. This is likely due to the fact that alpha-chymotrypsinogen A has a higher area of exposed hydrophobic segments than trypsinogen does. This observation also implies that as temperature increases, the interaction mechanism of alpha-chymotrypsinogen A with butyl-Sepharose changes from an adsorption-dominated process to a partitioning process. In addition, for octyl-Sepharose, the DeltaH value of alpha-chymotrypsinogen A is positive and decreases with temperature increment. However, the DeltaH value of trypsinogen was positive and increased with temperature. Therefore, we conclude that as temperature increases, the interaction mechanism of the proteins for octyl-Sepharose is a partitioning-dominated process. Copyright 2000 Academic Press.

Journal Article↗
PubMed2002

1pK and 2pK protonation models in the theoretical description of simple ion adsorption at the oxide/electrolyte interface: the analysis of temperature dependence of potentiometric titration curves.

Two models of oxide surface charging (1pK and 2pK) were used to describe the potentiometric titration curves measured by Blesa et al. (J. Colloid Interface Sci.101, 410 (1984)) at three temperatures and at three concentrations of electrolyte. Rudzinski et al. (Langmuir13, 483 (1997)) have applied the 2pK Triple Layer Model to analyze the above system earlier. Two calculation procedures based on the 1pK Basic Stern Model were developed to described Blesa's data. Since the experimental curves have the CIP (common intersection point) it was assumed that heat of electrolyte ion adsorption was equal to zero. We assumed two different values of the double layer innermost capacitance on both sides of PZC (point of zero charge), which was followed from asymmetry of surface charge curves relative to PZC. Moreover, it was necessary to take into consideration the dependence of capacitance on ionic strength and temperature. The quality of fit given by two models was comparable. Since the 1pK BSM is simpler than the 2pK TLM and includes not so many best-fit parameters it seems to be a better choice in this case. Discussion of the results obtained by other authors concerning the subject under consideration is also presented.

Journal Article↗
PubMed1996

Titration properties and thermodynamics of the transition state for folding: comparison of two-state and multi-state folding pathways.

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (Dphys or I). CI2 folds from a largely unstructured denatured state under physiological conditions via a transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (Dphys-->++) is very similar for both proteins.

Hydrogen-Ion Concentration↗
PubMed1999

Protein titration in the crystal state.

Proteins are complex structures whose overall stability critically depends on a delicate balance of numerous interactions of similar strength, which are markedly influenced by their environment. Here, we present an analysis of the effect of pH on a protein structure in the crystalline state using RNase A as a model system. By altering only one physico-chemical parameter in a controlled manner, we are able to quantify the structural changes induced in the protein. Atomic resolution X-ray diffraction data were collected for crystals at six pH* values ranging from 5.2 to 8.8, and the six independently refined structures reveal subtle, albeit well-defined variations directly related to the pH titration of the protein. The deprotonation of the catalytic His12 residue is clearly evident in the electron density maps, confirming the reaction mechanism proposed by earlier enzymatic and structural studies. The concerted structural changes observed in the regions remote from the active-site point to an adaptation of the protein structure to the changes in the physico-chemical environment. Analysis of the stereochemistry of the six structures provided accurate estimates of p Kavalues of most of the histidine residues. This study gives further evidence for the advantage of atomic resolution X-ray crystallographic analyses for revealing small but significant structural changes which provide clues to the function of a biological macromolecule.

Binding Sites↗
PubMed2001

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics.

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH<m-OCH3, p-OH<m-OH<m-OH, p-OCH3<bis-m-OH. The association constants K(a) are three to 11 times larger for the AATT site than for poly[d(A-T)]. The AATT site binds m-OH Hoechst with K(a)=3.8 x 10(9 )M(-1) and bis-m-OH Hoechst with K(a)=1.9 x 10(10 )M(-1), which are seven and 37 times higher than p-OH Hoechst (Hoechst 33258), respectively. The high K(a )values determined at equilibrium agree with the kinetically defined association constants K(kin)=k(on)/k(off). The association-rate parameters k(on) were obtained by stopped-flow kinetics and the dissociation-rate parameters k(off) by dissociation kinetics using poly[d(A-5BrU)]. For binding to the AATT site, k(on) values are similar and nearly diffusion-controlled (2.0 x 10(8) M(-1) x s(-1) to 2.9 x 10(8) M(-1) x s(-1)), while k(off) values (0.42 s(-1) to 0.012 s(-1)) depend on the phenyl substitution and determine the affinity. At the AATT site, the longest-living complex is formed when the dye carries a bis-m-OH phenyl group that probably integrates in a hydrogen-bonding network of water molecules. With poly(dA).poly(dT), poly[d(A-T)] and poly[d(A-5BrU)], k(on) (between 6.1 x 10(7) M(-1) x s(-1) and 5.2 x 10(8) M(-1) x s(-1)) depends on the DNA.

AT Rich Sequence↗
PubMed1998

The cardiac content of sarcoplasmic reticulum in the rat determined by calcium uptake rate, calcium oxalate capacity, ryanodine binding and thapsigargin titration.

The amount of cardiac sarcoplasmic reticulum in rat hearts was estimated by comparing marker activities in the isolated SR fraction with their activities in the homogenate. Four distinguishable markers were measured: the oxalate-supported rate of calcium uptake, the calcium oxalate capacity, 3H-ryanodine binding and the thapsigargin equivalents. The calcium uptake rate and capacity and thapsigargin equivalents were determined in the presence and absence of SR Ca2+ channel blockade with high concentrations of ryanodine. All of these activities are believed to be located only in the SR. However, the calculation of the heart content of SR was somewhat different for the four markers. The calcium uptake rate gave 8.4 mg SR protein per g tissue in the absence of ryanodine, and 9.6 mg per g in its presence; calcium oxalate capacity gave similar numbers, 9.9 mg per g in the absence of ryanodine and 8.0 mg per g in its presence. The thapsigargin titration gave similar equivalent with or without ryanodine, indicating that the homogenate contained about 8.0 mg of SR per g tissue. Using 3H-ranodine binding as a marker, the cardiac content of SR was calculated to be 16.7 mg per g. These differences are attributed to the non-ideal behavior of these markers. Some of the Ca2+ uptake activity is not thapsigargin sensitive, and some of the 3H-ryanodine binding does not fractionate with the SR Ca2+ uptake activity.

Animals↗
PubMed2001

Titration of low K(d) binding sites: binding of arginine analogs to nitric oxide synthases.

Spectrophotometrically monitored ligand titration is an important method for the determination of equilibrium dissociation constants (K(d)) from nitric oxide synthases (NOS). Low K(d) sites such as the tetrahydrobiopterin and arginine binding sites present difficulties in that experiments often require enzyme concentrations of the same magnitude as the K(d). An analytical method based on computer simulation is described that allows the estimation of K(d) values without an independent means of monitoring free ligand or without an accurate prior determination of the number of binding sites. The K(d) for arginine is approximately 0.5 microM for the tetrahydrobiopterin replete neuronal and inducible isoforms (nNOS and iNOS), while the endothelial isoform has a slightly higher K(d) (1.5 microM). N-OH-arginine (an intermediate) binds to nNOS with a K(d) of around 0.2 microM, while the inhibitors N-methyl-arginine and N-nitro-arginine bind more tightly; our best K(d) estimates are 100 nM or lower.

Animals↗
PubMed1994

Estrogen- and progesterone receptors in normal cycling endometrium as studied by end-point titration.

A thorough knowledge of the normal physiological fluctuations in estrogen- (ER) and progesterone receptors (PgR) is essential to characterize the changes in ER and PgR in the abnormal endometrium. We investigated the distribution of ER and PgR in frozen human cycling endometrial tissue using the commercially available ER- and PgR-ICA kits. Two-fold end-point titration (EPT) of ER and PgR antibodies was implemented to semi-quantitate more accurately ER and PgR. Semiquantitation of ER and PgR using EPT was significantly correlated to results obtained using either simple scoring or enzyme-immunoassay (EIA) methods. ER and PgR staining fluctuated in relation to the menstrual cycle. In most subphases PgR exceeded ER in both epithelial and stromal cells. Highest levels of ER and PgR were demonstrated in the glands of the functionalis in mid-to-late proliferative phases, whereas both receptors were almost undetectable by immunohistology in the glands of mid-to-late secretory phases. Endometrial stromal cells had high and nearly constant EPT values for PgR, but low values for ER throughout the menstrual cycle. EPT values for ER and PgR were generally higher in the basalis than in the functionalis but showed similar cyclic fluctuations. Our results further substantiate the view that the response to hormonal stimulation is cell-type specific, and suggest differences in steroid metabolism according to cell type and layer.

Adult↗
PubMed1980

Nalorphine's effect under several schedules of electric shock titration.

The effects of nalorphine were examined under a shock titration procedure in which squirrel monkeys received shock during 15-s period. If the monkey did not make five responses during the 15-s shock period, the shock was increased by one increment and maintained for another 15-s period. If five responses were made during the 15-s shock period, the shock was immediately terminated for 15s during which the chamber was dark (time out). After the 15-s time out period, the shock resumed at the next lower intensity. Three schedules of shock increment were examined in which shock increased from 0 to either 1.0, 2.0, or 5.5 mA. Under all three schedules of shock increment nalorphine decreased median shock levels and increased rates of responding in the presence of shock and in the absence of shock (i.e., during time out). These effects were apparent during hour 1 of the session. Responding during hours 2 and 3 of the session was unchanged.

Animals↗
PubMed1983

Electric shock titration in squirrel monkeys following administration of nantradol, levonantradol, or N-methyl levonantradol.

The effects of some cannabinoid-related analgesics were examined in squirrel monkeys responding under a schedule of shock titration. Nantradol (0.1-0.3 mg/kg), levonantradol (0.03-0.1 mg/kg), or N-methyl levonantradol (0.01-0.03 mg/kg) increased the level at which shock was maintained, but did not change rates of responding. Nantradol effects were not blocked by naloxone (0.3-3.0 mg/kg), but they were attenuated following daily administration of nantradol.

Analgesics↗