Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat: variation of the equilibrium and reverse rate constants with pH

We have determined for the first time the equilibrium constant, Keq, for the reaction of Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate), with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat. In the pH range 5.6 to 9.0 Kequ varies over four orders of magnitude--between ca 10 and 10(-3)--for all hemoglobin derivatives. Using these Kequ values and published data on the dependence of the apparent second order forward rate constant, kf, on pH we have calculated the apparent second order reverse rate constant, kr, as a function of pH. This parameter increases strongly with pH, particularly above pH 7.5. Quantitative analyses of the pH dependence profiles of log10kr indicate that the reverse reaction is coupled to the ionization of two groups on the protein with pKas of 7.2+/-0.2 and 9.4+/-0.1 in the major hemoglobin and 6.7+/-0.3 and 8.4+/-0.1 in the minor hemoglobin.     

Transition of hemoglobin between two tertiary conformations: determination of equilibrium and thermodynamic parameters from the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group

The equilibrium constant of the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of hemoglobin decreases by 2 to 3 orders of magnitude between pH 5.6 and 9. The reaction is coupled to the ionizations of two groups on the protein. At 25 degrees C one group has a pK(a) of 5.31+/-0.2 when hemoglobin is in its (tertiary) r conformation, typified by the thiolate anion form of CysF9[93]beta; this changes to 7.73+/-0.4 in the (tertiary) t conformation, typified by the mixed disulfide form of the sulfhydryl. The second group ionizes with a pK(a) of 7.11+/-0.4 in the r conformation; this changes to 8.38+/-0.2 in the t conformation. K(rt), the equilibrium constant for the r<-->t isomerization process, is 0.22+/-0.06. The standard enthalpy and entropy changes for the isomerization are DeltaH(o)(rt)=24.2 kJ mol(-1) and DeltaS(o)(rt)=68.8 JK(-1)mol(-1), respectively.     

Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica)

We demonstrate that 5,5'-dithiobis(2-nitrobenzoate) - DTNB - reacts with only CysF9[93]beta and CysB5[23]beta among the multiple sulfhydryl groups of the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica). K(equ), the equilibrium constant for the reaction, does not differ very significantly between the two hemoglobins. It decreases 430-fold between pH approximately 5.6 and pH approximately 9: from a mean of 7+/-1 to a mean of 0.016+/-0.003. Quantitative analyses of the K(equ) data based on published X-ray and temperature-jump evidence for a tertiary structure transition in liganded hemoglobin enable the calculation of K(rt), the equilibrium constant for the r<---->t tertiary structure transition. K(rt) differs significantly between the two hemoglobins: 0.744+/-0.04 for the major, 0.401+/-0.01 for the minor hemoglobin. The mean pK(a)s of the two groups whose ionizations are coupled to the DTNB reaction are about the same as previously reported for mammalian hemoglobins.     

Temperature-jump studies on hemoglobin. Kinetic evidence for a non-quaternary isomerization process in deoxy- and carbonmonoxyhemoglobin

Temperature jumps on mixtures of hemoglobin and pH indicators give rise to relaxation signals in the microsecond range. The pH and concentration dependences of the reciprocal relaxation time, 1/tau, may be rationalized on the basis of a reaction scheme in which a slow isomerization process in the protein moiety is coupled to a rapid co-operative ionization of two protons. At 11 degrees C the rate constants of the isomerization are kr = 4.2(+/- 1.8) x 10(4) s-1 and kf = 1.3(+/- 0.1) x 10(4) s-1 in deoxyhemoglobin; in carbonmonoxyhemoglobin they are kr = 3.9(+/- 1.3) x 10(4) s-1 and kf = 5.3(+/- 1.8) x 10(3) s-1. The pKa values of the coupled ionizing groups are 5.3 in deoxy- and 6.0 in carbonmonoxyhemoglobin. Modification of the CysF9(93) beta sulfhydryl group with iodoacetamide abolishes the pH dependence of 1/tau, suggesting that this sulfhydryl is involved in the isomerization process. Consideration of the X-ray structure of oxyhemoglobin allows a structural interpretation of the results. It is concluded that the isomerization may be important for the physiological function of hemoglobin, but that it is not identical with the quaternary structure transition.     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

Varying apparent rate constant: determination of uptake and release of protons during tetramer-dimer dissociation in human hemoglobin A

The effect of association-dissociation on the sulphydryl reactivity of human hemoglobin A is reported. The reactivity of CysF9(93)beta towards the sulphydryl reagent, 5,5'-dithiobis(2-nitrobenzoate), is higher at lower concentrations of hemoglobin at all pH values. This is because hemoglobin dimers have higher sulphydryl reactivity than tetramers and it is known that the proportion of dimers increases as the hemoglobin concentration decreases. This study takes advantage of this observation to determine the tetramer-dimer dissociation constant, K(4,2), of hemoglobin A and subsequently the proton uptake and the proton release during this process. The concentration dependence profiles of the apparent second-order rate constants, k(app), show that (between 2 and 20 microM heme) k(app) decreases with increasing hemoglobin concentration. Above 30 M heme k(app) remains fairly constant for all hemoglobin derivatives (oxy, carbonmonoxy and aquomethemoglobin) used. The pH dependence of the negative logarithm of tetramer-dimer dissociation constant, pK(4,2), for oxy- (and for carbonmonoxy-) hemoglobin exhibits a biphasic character with a maximum near pH 7.4 (and 6.6). For aquomethemoglobin, pK(4,20 decreases with increasing pH. The tetramer-dimer dissociation of human oxyhemoglobin A at an ionic strength of 200 mM uptakes 0.87 +/- 0.09 mole of protons between pH 6.2 to 7.4 phase and releases 0.84 0.09 mole of protons between pH 7.4 and 9.0 phase. Under a similar condition carbonmonoxyhemoglobin uptakes 0.54 +/- 0.05 mole of protons between pH 5.8 and 6.6 phase and releases 0.48 +/- 0.05 mole of protons between pH 6.6 and 9.0 phase. Aquomethemoglobin has only a single phase, it releases 0.39 +/- 0.05 mole of protons during tetramer-dimer dissociation.     

Ionizable groups linked to the reaction of 2,2'-dithiobispyridine with hemoglobin.

The pH-dependence of the second-order rate-constant for the reaction of 2,2'-dithiobispyridine with the CysF9(93) beta sulphydryl group of hemoglobin in the R quaternary structure is analyzed in terms of a tentative model based on the observation that this sulphydryl exists as a mixture of two tertiary conformations in dynamic equilibrium. For the four aquomethemoglobins studied (human A and S, dog and rabbit), the equation derived from this model gives a better fit than a simpler equation based on the assumption of only one tertiary conformation. For the corresponding carbonmonoxyhemoglobins the simpler equation gives a better fit. The dog and rabbit oxy and azidomet data are better fitted by the model equation, whereas the data for the corresponding human A and S derivatives are better fitted by the simpler equation. From the analysis pKa values of 6.1 and 8.7 are obtained for the ionization of groups coupled to the presumed conformational transition. The pKa of 6.1 is assigned to HisHC3(146) beta; the pKa of 8.7 is assigned to the CysF9(93) beta sulphydryl group in its external conformation. It is estimated that the pKa of this sulphydryl may be as high as 12.9 in its internal conformation.     

Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits

The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits. Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase. Labeling with [14C]iodoacetamide and [14C]N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively. The epsilon subunit was not labeled. Application of the method of Creighton [Nature (London) (1980) 284, 487-489] showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively. This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated. The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes [H. Kanazawa, T. Kayano, K. Mabuchi, and M. Futai (1981) Biochem. Biophys. Res. Commun. 103, 604-612; N. J. Gay and J. E. Walker (1981) Nucl. Acids Res. 9, 2187-2194] where three cysteinyl residues/alpha subunit have been found. It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.     

Chemical synthesis of human beta-endorphin(1-27) analogs by peptide segment coupling. Leucine and glycine residues bearing thiocarboxyl functions as junctions for peptide segment coupling.

[Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2, two analogs of human beta-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides. Msc-[Gly8]beta hEP(1-8)SH and Msc-[L-Leu8]beta hEP(1-8)SH, were synthesized by standard solid phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[alpha-(Boc-L-Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 in the segment coupling reaction were approximately 18%. Less than 1% of racemization of Leu-8 occurred during coupling of Msc-[L-Leu8]beta hEP(1-8)SH to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of beta-endorphin(1-27) and its analogs influenced the receptor binding activity.     

Crystallographic analysis of the interaction of nitric oxide with quaternary-T human hemoglobin

In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the alpha-subunits but not the beta-subunits. This finding supports Perutz's theory that ligand binding induces tension in the alpha Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin betaW37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligand-induced movement of the alpha-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the beta- and the alpha-subunits remain intact in crystalline betaW37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9)beta has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9)beta disrupts the intrasubunit salt bridge between His146(HC3)beta and Asp94(FG1)beta, a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93(F9)beta that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N.-O-H radical intermediate and not Hb-S-N=O as previously suggested.     

Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp

Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variability in the pKa of histidine side-chains correlates with burial within proteins

Acidic pKas of histidines buried within the protein interior are frequently rationalized on the contradictory basis of either polar interactions within the protein or the effects of a hydrophobic environment. To examine these relationships, we surveyed the buried surface area, depth of burial, polar interactions, and crystallographic temperature factors of histidines of known pKa. It has been found that buried environments of histidines do not always result in acidic pKas. Instead, the variability of histidine pKas increases for residues where the majority of the side-chain is buried. Because buried histidines are always found in mixed polar/apolar environments, multiple environmental contributions to pKa values must be considered. However, the quantitative relationships between heterogeneous environments and pKa values are not immediately apparent from the available data.     

A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [3H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 +/- 0.017% of hemoglobin in blood of normal adult, 0.11 +/- 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [3H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.     

Hemoglobins with multiple reactive sulphydryl groups: The reaction of dog hemoglobin with 5,5′-dithiobis (2-nitrobenzoate)

Dog hemoglobin has four sulphydryl groups per (tetramer) molecule located at the G18(111)a and F9(93)β positions. The two sulphydryls at the G18(111)a positions are unreactive toward nonmercurial sulphydryl reagents, but those at the F9(93)β positions are reactive toward these reagents. We have studied the kinetics of the reaction of dog hemoglobin with 5,5′-dithiobis (2-nitrobenzoic acid) as a function ofpH. At allpH values studied, the reaction is kinetically monophasic. Quantitative analysis of thepH dependence of the apparent second-order rate constant shows that two ionizable groups are linked to the reaction of the sulphydryl group. TheirpK _a values are 5.57 and 9.0. These values are assigned to HisHC3(146)β and to the CysF9(93)β sulphydryl. We find that dog carbonmonoxyhemoglobin is significantly—almost an order of magnitude—less reactive than the aquomet, azidomet, and oxy derivatives. This result may be due to a greater tendency (at acidpH) for the salt bridge between HisHC3(146)β and AspFG1(94)β to form in the carbonmonoxy than in the other derivatives. Formation of this salt bridge is known to hinder access to the CysF9(93)β sulphydryl [Perutz, M. F. (1970),Nature 228, 734–739].     

Human μ—opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi／0 proteins

Human mu-opioid receptor (HmuOR) with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells. The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by mu-selective agonists [D-Ala2, N-methyl-Phe4, glyol5]enkephalin (DAGO), Ohm, and morphine, but neither by delta nor by kappa selective agonist. Na+ (100 mM) and GTP (50 microM) could reduce HmuOR agonists etorphine and Ohm affinity binding to the overexpressed HmuOR. mu-selective agonists DAGO and Ohm effectively stimulated [35S]GTP-gammaS binding (EC50 = 2.7 nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX). These results demonstrated that HmuOR overexpressed in Sf9 insect cells functionally coupled to endogenous G(i/o) proteins.     

In vitro study of buffer capacity of blood as a function of hemoglobin concentration in rabbits]

Rabbit blood samples with different hemoglobin contents were equilibrated with 3,5 and 7% of CO2 in air permetting buffer line determination. Normal buffer line was --24.5 +/- 1.45 Slyke (M +/- 1 SE). The buffer line/hemoglobin concentration calculated regression line was beta = 2.46 [Hb] + 6.56. These data are compared with results obtained in human blood.     

Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides

The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.     

Interaction of a spin-labeled phenylalanine analogue with normal and sickle hemoglobins: detection of site-specific interactions through spin-label-induced 1H NMR relaxation

In a preliminary report, we have previously shown that N-[(2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)carbonyl]-L-phenyl ala nine tert-butyl ester (SL-Phe) exhibits specific binding to hemoglobin and an antiaggregation activity more than 2 orders of magnitude greater than that of phenylalanine [Lu, H.-Z., Currie, B. L., & Johnson, M. E. (1984) FEBS Lett. 173, 259-263]. Transverse 1H NMR relaxation measurements have been used to investigate the interaction of SL-Phe with hemoglobin molecules by use of the resonances assigned to the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 or beta 97 His residues as intrinsic probes. Distance calculations using the paramagnetically induced relaxation data suggest that the SL-Phe binding site is approximately 12-16 A away from the C2 protons of the beta 2 His and the beta 146 or beta 97 His residues in the (carbonmonoxy)hemoglobin tetramer; for deoxyhemoglobin, the distances are approximately 14-17 A between the SL-Phe binding site and the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 His residues. Calculations using the (carbonmonoxy)hemoglobin crystal atomic coordinates only restrict the probable SL-Phe binding region to the full F and H helices of the beta-chain and a small section of the alpha-chain. For deoxyhemoglobin, the distance calculations provide greater restrictions on the probable binding region, limiting it to small sections of the beta-chain F, G, and H helices near the EF bend and to a few residues on the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP: iodination and binding characteristics of a vasopressin receptor ligand

A radioiodinated vasopressin antagonist, d(CH2)5[Tyr(NH2)9]AVP has been prepared. Iodination was carried out at the phenyl moiety of the tyrosylamide residue at position 9, followed by HPLC purification. Non-radiolabelled monoiodinated antagonist was used as a reference for identification. 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP binding appeared to take place with a dissociation constant of 0.28 +/- 0.09 nM (Kd +/- SD) to V1 vasopressin receptors on rat liver membranes.