Interaction of fibrinogen-binding tetrapeptides with fibrin oligomers and fine fibrin clots

The polymerization of fibrin, at pH 8.5 and ionic strength 0.45, and under conditions where the action of thrombin on fibrinogen was the rate-determining step, was interrupted by inactivating thrombin with p-nitrophenyl-p′-guanidinobenzoate (NPGB). Addition of the tetrapeptide Gly-Pro-Arg-Pro (GPRP) partially dissociated the fibrin oligomers as shown by subsequent ligation with Factor XIIIa and calcium ion followed by denaturation and gel electrophoresis; polyacrylamide gel electrophoresis with reduction showed a decrease in the proportion of γ-γ ligation compared with controls untreated by GPRP, and agarose gel electrophoresis showed a shift in the distribution of oligomer sizes. The dissociation was accomplished within 15 min and its extent was consistent with establishment of an equilibrium in which two molecules of GPRP react to sever an oligomer. When GPRP was introduced into fine unligated fibrin clots by diffusion, there was some dissociation as shown by differences in the degree of γ-γ ligation after treatment by Factor XIIIa; but the action of GPRP was much slower and less complete than on soluble oligomers. However, even a small amount of dissociation affected the mechanical properties of fine clots profoundly. The shear modulus (measured 25 s after application of stress) decreased progressively with increasing concentration of GPRP introduced by diffusion. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. If the steadystate creep rate is interpreted in terms of an effective viscosity, the latter is decreased by up to three orders of magnitude by the presence of GPRP. In terms of transient network theories of viscoelasticity, the average lifetime of a network strand is greatly diminished. However, the total density of strands remains constant during creep and creep recovery as shown by constancy of the differential modulus or compliance. Removal of GPRP by diffusion only partially restores the original shear modulus and creep behavior of the original clot. Some limited data on the effect of the tetrapeptide Gly-His-Arg-Pro are also reported.     

Interaction of the fibrinogen-binding tetrapeptide Gly-Pro-Arg-Pro with fine clots and oligomers of alpha-fibrin; comparisons with alpha beta-fibrin

The tetrapeptide Gly-Pro-Arg-Pro(GPRP) was introduced by diffusion into fine unligated clots formed from human fibrinogen at pH 8.5 and ionic strength 0.45 by batroxobin (αβ-fibrin) and by thrombin (α-fibrin). The α-fibrin clots were essentially liquefied at GPRP concentrations above 1 mM and αβ-fibrin clots above 15 mM, and the degree of polymerization of the resulting oligomers decreased progressively with increasing GPRP concentration as shown by γ-γ ligation with factor XIIIa and subsequent gel electrophoresis. Much smaller concentrations of GPRP, when introduced into unligated clots by diffusion, were sufficient to modify their mechanical properties profoundly. The shear modulus of elasticity G₂₅ measured 25 s after imposition of stress fell, for example, by a factor of 0.4 at 0.1 mM GPRP in α-fibrin and at 1.1 mM in αβ-fibrin. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. This behavior, and the corresponding decrease in steady flow viscosity, may be interpreted in terms of competition of GPRP with A sites on the E domains of fibrin monomers for bidning to “a” sites on the D domains, resulting in a moderate increase with increasing GPRP concentration of the average proportion of severed network strands and an enormous increase in the rate at which all strands dissociate and reassociate. Reassociation of severed strands in new configurations is a necessary corollary since the differential modulus or compliance remains constant during creep and creep recovery. The greater susceptibility of α-fibrin clots to interaction with GPRP is attributed to stabilization of contacts between monomer units by Bb associations in αβ-fibrin. Ligated clots, with or without GPRP, exhibited essentially no time-dependent creep and no irrecoverable deformation, corresponding to an absence of any severance of network strands.     

Acid-base properties of thymopoietin-type tri- and tetrapeptides and their derivatives.

The submolecular basicities of 21 immuno-modulating, thymopoietin-type di-, tri-, and tetrapeptides were studied and characterized in terms of group constants and partial microconstants. All compounds were derivatives of the H-Arg-Lys-Asp-OH tripeptide. Modifications within four covalent bonds of the basic site (esterification, acylation, curtailment or addition at C-terminal end, exchange of amino acids) cause significant changes in the scheme of protonation and in the individual basicity of proton binding sites. Configurational changes of the component amino acids, however, do not cause significantly different basicities in the diastereomers.     

Electrical properties of hornet silk: temperature dependence

Hornet silk is a polymer of amino acids. One of the known properties of polymers is their electrical activity. The present study describes the results of electrical measurements carried out vertically on the silk cap of pupae of the Oriental hornet Vespa orientalis (Hymenoptera, Vespinae). The measurements undertaken were the temperature-dependent electric current, voltage and resistance, all measured within the range of biological temperatures, as well as the capacitance. The temperature-dependent spontaneous current attained values up to 327 nano Amperes (nA) while the maximal voltage reached 347 millivolt (mV). The electrical resistance was low and steady (1-20 mu omega) at temperatures ranging between 19-32 degrees C, but at lower or higher temperature it increased fairly sharply by about three orders of magnitude. The electrical capacitance, computed according to the discharge curve (decay curve) amounted to 0.4 microFarad (microF). The paper also discusses the role of the pupal silk as producer of a 'clean room' while the cuticle is being laid down by the pupae after undergoing metamorphosis, as well as the significance of the measured electrical parameters vis-à-vis the developing pupae.     

pH dependence of the interaction of hirudin with thrombin.

The kinetics of the inhibition of human alpha-thrombin by recombinant hirudin have been studied over the pH range from 6 to 10. The association rate constant for hirudin did not vary significantly over this pH range. The dissociation constant of hirudin depended on the ionization state of groups with pKa values of about 7.1, 8.4, and 9.2. Optimal binding of hirudin to thrombin occurred when the groups with pKa values of 8.4 and 9.0 were protonated and the other group with a pKa of 7.1 was deprotonated. The pH kinetics of genetically engineered forms of hirudin were examined in an attempt to assign these pKa values to particular groups. By using this approach, it was possible to show that protonation His51 and ionization of acidic residues in the C-terminal region of hirudin were not responsible for the observed pKa values. In contrast, the pKa value of 8.4 was not observed when a form of hirudin with an acetylated alpha-amino group was examined, and, thus, this pKa value was assigned to the alpha-amino group of hirudin. The requirement for this group to be protonated for optimal binding to thrombin is discussed in terms of the crystal structure of the thrombin-hirudin complex. Examination of this structure allowed the other pKa values of 7.1 and 9.2 to be tentatively attributed to His57 and the alpha-amino group of Ile16 of thrombin.     

The pH and temperature dependence of the interaction of steroid hormones with the transport system of glucose in human erythrocytes.

The thermodynamic parameters deltaH degrees, deltaF degrees, deltaS degrees and the pH dependence of the interaction between steroids and the glucose carrier in human erythrocytes have been studied. The results indicate that, according to the structure of the steroids, hydrophilic as well as hydrophobic bonds can be involved in the association. For the binding of the C-21-steroids to the carrier the polarity rule has been observed to be valid. On the basis of the thermodynamic parameters it has been found that in the association process of the steroids with the carrier, simultaneous randomization of ordered water molecules is important.     

Temperature and the formation of radiation-induced chromosome aberrations. II. The temperature dependence of lesion repair and lesion interaction

Dicentric chromosome aberration yields have been measured after single-exposure and split-dose irradiations of human lymphocytes with 150 kV X-rays. Various temperature programmes between 4 and 37 degrees C were applied before, during and after irradiations and in the radiation-free interval. It was found that chromatin lesion repair was completely suppressed at 21 degrees C and below, whereas lesion formation is reduced only below 17 degrees C. The interaction between repairable lesions which leads to exchange-type aberrations is also suppressed by low temperatures. Hypothermic suppression of chromatin lesion repair and interaction is fully reversible at least up to 12 h of maintenance of the 'stored' state of these lesions.     

pH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies.

The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG.     

Spin label method reveals barnase-barstar interaction: a temperature and viscosity dependence approach

Spin labeling was used to study the protein-protein interaction between the enzyme barnase (Bn) and its inhibitor barstar (Bs). A mutant of barstar (C40A), which contains only one cysteine, C82, located near the Bn-Bs contact region, was selectively modified by two spin labels having different lengths and structures of the flexible tether. The formation of a strong protein complex resulted in significant restriction of spin label mobility at the C82 residue of barstar, as indicated by notable changes in the recorded EPR spectra. The dependence of the separation between broad outer peaks of the EPR spectra on viscosity at constant temperature was used to evaluate the order parameter S and the rotational correlation time tau (a temperature-viscosity dependence approach). The order parameter S, which characterizes fast reorientation of a spin label relative to the protein molecule, sharply increases and approaches unity when Bs binds to Bn. In addition, formation of a Bs-Bs complex was observed; it is also accompanied by restriction of spin label mobility. At the same time, the rotational correlation times tau of spin-labeled Bs, its complex with Bn, and the Bs dimer in solution agree well with their molecular masses. This indicates that barstar, its complex with barnase, and barstar dimer are rigid protein entities. The described approach is suitable for studying any spin-labeled macromolecular complexes.     

Copper(II) interaction with tetrapeptides containing proline and phenylalanine: A potentiometric and spectrophotometric study

The synthesis of four tetrapeptides, L-Phe-L-Pro-Gly-Gly, Gly-L-Pro-L-Phe-Gly, Gly-L-Pro-D-Phe-Gly, and Gly-L-Pro-Gly-L-Phe is described. The hydrogen ion and copper(II) complex formation constants have been measured at 25°C and I = 0.10 mol dm^?3 (KNO₃). Circular dichroism spectra have been recorded for copper(II)-peptide mixtures as a function of pH. The potentiometric and Spectrophotometric studies have been combined to ascertain the complex species over a broad pH range. The results obtained support the earlier suggestion on the specific role of a proline residue as a “break-point” in copper complex formation with peptides: the insertion of a proline residue into the second position of a tetrapeptide sequence leads to a novel coordination mode in Cu(II)-tetrapeptide systems.     

A comparative study of the temperature dependence of the oxygen-binding properties of mammalian hemoglobins.

The effect of temperature on the oxygen-binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3-diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy]. Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.     

Rate enhancement specificity with alpha-chymotrypsin: temperature dependence of deacylation.

The relative rate of the hydrolysis of 2-(5-n-alkyl)furoyl-alpha-chymotrypsin reaches a maximum with the propyl derivative. The Arrhenius plots for the hydrolyses of the 2-furoyl-, 2-(5-ethyl)furoyl-, and 2-(5-n-propyl)furoyl-alpha-chymotrypsins display a discontinuity, while the plots obtained with the ramaining furoyl derivatives 5-methyl, 5-n-butyl, and 5-n-amyl are linear. We conclude that the deacylation of the furoyl derivatives of alpha-chymotrypsin involves a minimum of two elementary reaction steps. Depending upon the reaction conditions, rate enhancement specificity appears to be either entropy or enthalpy controlled.     

The temperature dependence of some kinetic and conductance properties of acetylcholine receptor channels

We examined the temperature dependence of single-channel properties of the nicotinic acetylcholine receptor channel from clonal BC3H-1 cells over a range of 10-40 degrees C. We found temperature sensitivities (Q10 values) of 2-4 for the mean channel open time. The Q10 did not depend strongly on voltage and the voltage dependence of the mean open time was temperature-independent. The Q10 of closing rate of the long-lived open state was 3-4 but the Q10 of closing rate of the brief open state was independent of temperature. The duration of brief closures could be measured only between 10 and 25 degrees C. Since this approached the limit of the experimental time resolution, an accurate determination of the Q10 could not be made. The current decay due to desensitization after rapid application of high concentrations of agonist varied with a Q10 of about 2. The conductance of single channels (the inverse of the ion translocation rate) had a Q10 of 1.3-1.5. We found no obvious nonlinearities in the Arrhenius curves for any of the measured properties.     

Nucleic acid interaction with VERO cells. A temperature barrier in the interaction pattern.

The interaction of VERO cell monolayers with spin (nitroxide)-(labeled polynucleotides (1(N)n) was examined by electron spin resonance (ESR) spectroscopy at various temperatures. Nitroxide labels covalently linked to (A)n, (dUfl)n, (U)n and (A)n . (U)n were used to monitor the interaction. The VERO cells were grown on small quartz plates with a cell viability of 95% or better and then used directly for the ESR studies. The ESR results indicated that the interaction between VERO cells and spin-labeled nucleic acids is temperature dependent. No temperature dependence was found when VERO cells were in contact with nitroxide radicals which were free in solution or covalently bound to Sepharose 4B. The temperature dependence established with nitroxide-labeled nucleic acids indicates that a temperature barrier must exist between 20 and 26 degrees C for the interaction between nucleic acids and VERO cells; namely, at 26 degrees C or above spin-labeled nucleic acids interact significantly with a VERO cell surface; whereas, at 20 degrees C the ESR signal reports no interaction. It is concluded that a temperature-dependent phase transition of membrane components or cell surface products active at 26 degrees C or above play a key role in the nucleic acid cell surface interaction process.