The conformational and dynamic basis for ligand binding reactivity in hemoglobin Ypsilanti (beta 99 asp-->Tyr): origin of the quaternary enhancement effect.

Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxygen with little or no cooperativity and with high affinity [Doyle, M. L., et al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81-91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new quaternary structure that partially resembles the recently described R2 structure [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very little is known about either the solution phase conformations of the liganded and deoxy forms of HbY or the molecular basis for the large quaternary enhancement effect (Doyle et al., 1992). In this study, near-IR absorption, Soret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are used to probe the liganded and deoxy derivatives of HbY in solution. Nanosecond time-resolved near-IR absorption measurements are used to expose the relaxation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebinding curves for photodissociated COHbY. The spectroscopic results indicate that COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha 1 beta 2 interface. The deoxy derivative of HbY has spectroscopic features that are very similar to those observed for species assigned to the deoxy R or half-liganded R conformations of human adult hemoglobin (HbA). The 10 ns to 100 micros relaxation properties of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observed. The near-absence of relaxation in the HbY photoproduct explains the differences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 1992). In addition, the spectroscopic data and the X-ray crystallographic results explain the slow relaxation for HbY and the near-absence of cooperative ligand binding for this protein based on the behavior of the penultimate tyrosines.

Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.

Functional implications of the proximal hydrogen-bonding network in myoglobin: a resonance Raman and kinetic study of Leu89, Ser92, His97, and F-helix swap mutants.

Resonance Raman spectra have been obtained for both the equilibrium deoxy derivative and the 10 ns photoproduct of the CO derivative of several mutants of sperm whale myoglobin. The particular mutations on the F-helix were chosen to expose the role of the proximal hydrogen-bonding network in maintaining the position of the heme, the proximal histidine, and the heme-7-propionate. In each mutant, one or more hydrogen bonds are altered or eliminated. A careful comparison of the spectra from the equilibrium and transient five coordinate species indicates that the tertiary relaxation after photodissociation is nearly complete within 10 ns, as is the case in the WT protein. The iron-proximal histidine stretching mode (nu(Fe-His)) and several low-frequency propionate-sensitive modes in the Raman spectra reveal the impact of specific disruptions in the hydrogen-bonding network on the heme pocket geometry. Two categories of perturbation are observed with respect to nu(Fe-His): (1) a shift in the peak frequency without a change in line shape and (2) changes in the overall line shape which may or may not be accompanied by a frequency shift. The alterations in the nu(Fe-His) band are interpreted as arising from conformational heterogeneity and local geometrical changes within the pocket, including movement of the heme group, and are discussed in terms of changes in the population distribution as revealed via a curve-fitting analysis. None of the frequency shifts in the nu(Fe-His) band are as large as that reported for the His93Gly(imidazole) mutant, suggesting that the covalent linkage between the heme and His93 plays a crucial role in maintaining the geometry of the proximal pocket. Molecular modeling indicates that the nu(Fe-His) frequency shifts observed in the present study originate from changes in the His93 imidazole ring azimuthal angle. The systematic variations in the interactions of the heme-7-propionate in the mutants have exposed several properties of the propionate-sensitive Raman bands. The frequencies of nu9 (the 240 cm-1 shoulder on the nu(Fe-His) band) and delta(cbetacccd) at approximately 370 cm-1 appear to be correlated. A decrease in hydrogen-bond strength to this propionate in response to changes in stereochemistry or degree of disorder is associated with a decrease in the frequency of both nu9 and delta(cbetacccd). The mutations that cause a weakening of the hydrogen bonding to the heme-7-propionate also result in changes in nu(Fe-His) which are interpreted as evidence that this propionate participates in the anchoring of the heme within the heme pocket. Changes in gamma7 at approximately 300 cm-1, gamma6 at approximately 335 cm-1, and nu8 at approximately 342 cm-1 are discussed in terms of pocket disorder. A titration from pH 5.1 to 7.4 suggests that His97 is protonated in the WT protein by pH 5.1. Geminate-rebinding studies on these mutants indicate that disruption of the hydrogen-bonding network has only modest effects on ligand-binding kinetics, suggesting that the role of the hydrogen-bonding network may be one of maintaining heme pocket stability rather than of specific protein function.

A possible allosteric communication pathway identified through a resonance Raman study of four beta37 mutants of human hemoglobin A.

The highly conserved tryptophan at position beta37 occupies a key locus at the hinge region within the alpha1beta2 interface of the mammalian hemoglobins. This residue is thought to play an important role in mediating the heme-heme interaction associated with the cooperative binding of oxygen; however, its explicit function is unclear. In this study, the proximal heme environments of several beta37 mutants of adult human hemoglobin (HbA) are probed using visible (Soret band enhanced) resonance Raman spectroscopy. In the equilibrium deoxy derivatives of these mutants, a systematic variation in proximal strain, as reflected in the iron-proximal histidine (F8) stretching frequency, nu(Fe-His), is seen upon mutation of the beta37 residue. The variation in proximal strain correlates with both the ligand binding rates [Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335] and conformational changes observed at the FG corner through X-ray crystallography [Kavanaugh et al. (1998) Biochemistry 37, 4358-4373]. The results from the deoxy samples indicate a plasticity of the tertiary structure within the T quaternary state. The correlation between the X-ray data and the Raman supports the idea that the proximal strain at the heme within the T state can be modulated by a combination of forces including those arising from the hinge region of the alpha1beta2 interface, from the binding of allosteric effectors, and from the degree of iron displacement from the heme plane. Each of these contributors appears to operate through a shifting of the F helix either away from or toward the FG corner. The Raman spectra obtained from the 10 ns CO photoproduct of the beta37 mutant Hb's indicate that these mutants contain an altered coupling between the R state alpha1beta2 interface and the proximal heme environment. This altered coupling could be due to either dissociation of the ligated mutant tetramers into dimers or the formation of an R state tetramer with significantly weakened hydrogen bonds and van der Waals contacts between the alpha1 and beta2 subunits at the interface. In either case, the results reveal a clear-cut structural basis for the quaternary enhancement effect in which the normal R state quaternary structure produces a higher affinity ligand binding site than that which occurs in the corresponding dimeric form of the protein. The normal R state interface is shown to be important for stabilizing a favorable ligand binding environment that persists long enough after laser photolysis to enhance the geminate rebinding process within the photoproduct. The addition of IHP to the solution of mutant COHb proteins results in photoproduct spectra that are all identical and are consistent with the ligand-bound derivatives having either a T state structure or a very strained and anomalous R state structure.

Preparation and kinetic characterization of a series of betaW37 variants of human hemoglobin A: evidence for high-affinity T quaternary structures.

Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.

A comparison of functional and structural consequences of the tyrosine B10 and glutamine E7 motifs in two invertebrate hemoglobins (Ascaris suum and Lucina pectinata).

The architecture of the distal heme pocket in hemoglobins and myoglobins can play an important role in controlling ligand binding dynamics. The size and polarity of the residues occupying the distal pocket may contribute steric and dielectric effects. In vertebrate systems, the distal pocket typically contains a "distal" histidine at position E7 and a leucine at position B10. There are several invertebrate organisms that have hemoglobins or myoglobins that display a pattern in which residues E7 and B10 are a glutamine and tyrosine, respectively. These proteins often have very high oxygen affinities stemming from very slow ligand off rates. In this study, two such hemoglobins, one from the nematode Ascaris suum and the other from the sulfide-fixing clam Lucina pectinata, are compared with respect to conformational and functional properties. Ultraviolet resonance Raman spectroscopy and visible resonance Raman spectroscopy are used to probe, respectively, the ligand-dependent hydrogen bonding pattern of the tyrosine residues and the proximal heme pocket interactions. Fourier transform infrared absorption spectroscopy is used to probe the dielectric properties of the distal heme pocket through the stretching frequency of carbon monoxide bound to the heme. Functionality is probed through the geminate rebinding of both CO and O2. The findings reveal two very different patterns indicative of two different mechanisms for achieving low oxygen off rates. In Hb Ascaris, a hydrogen bonding network that includes the E7 Gln, B10 Tyr, and oxygen bound to the heme results in a tight cage for the oxygen. Dissociation of the O2 requires a large amplitude conformational fluctuation that results both in a spontaneous dissociation of the oxygen through the loss of hydrogen bond stabilization and in an enhanced probability for ligand escape though the transient disruption and opening of the tight distal cage. In the case of the Hb from Lucina, there is no evidence for a tight cage. Instead the data support a model in which the hydrogen bonding network is far more tenuous and the equilibrium state of distal pocket is far more open and accessible than is the case in Ascaris. The results explain why Hb Ascaris has one of the highest oxygen affinities known (P50 approximately 10(-)3 Torr) while Hb Lucina II has an oxygen affinity comparable to that of Mb (P50 = 0.13 Torr) even though both of these Hbs contain the B10 Tyr and E7 Gln motif and display very low oxygen off rates. The roles of water and proximal strain are discussed.

Quaternary structure sensitive tyrosine interactions in hemoglobin: a UV resonance Raman study of the double mutant rHb (beta99Asp-->Asn, alpha42Tyr-->Asp).

Two interactions involving tyrosines have been implicated in the communication pathway that links ligand binding to quaternary state changes in hemoglobin. Tyr alpha(1)42 stabilizes the alpha1beta2 T state interface through the formation of a hydrogen bond to Asp beta(2)99. The side chains of the penultimate Tyr residues (alpha140 and beta145) occupy the pockets made by helicies F and H in the deoxy form with the phenolic hydroxyl hydrogen bonded to the carbonyl group of Val FG5. Early crystallographic studies indicated that in the R form the penultimate Tyr is expelled out of the pocket, thus eliminating the hydrogen bond. This hydrogen bond has been considered to play an important role in maintaining the low-oxygen-affinity state (T state) in deoxy HbA, but a later higher resolution crystallographic study (Shannon, 1983) failed to reveal such movement of this Tyr during the R --> T transition. Nevertheless, conversion of this Tyr to Phe increases oxygen affinity considerably, suggesting that hydrogen bonding is involved in oxygen affinity modulation. Earlier ultraviolet resonance Raman results reported by Spiro and co-workers [Rodgers et al. (1992) J. Am. Chem. Soc. 114, 3697-3709] were used to conclude that the significant quaternary structure dependent changes observed in tyrosine Raman bands are due to the formation of the T state hydrogen bond with Tyr alpha42 acting as a proton acceptor rather than being the anticipated proton donor, as would be expected if Asp beta99 were ionized. This surprising result rests on the assumption that changes in the environment of Tyr alpha42 are the overwhelming contributor to the R - T UV Raman difference spectrum. In this study, a cooperative double mutant lacking Tyr alpha42, [rHb (Asp beta99 --> Asn, Tyr alpha42 --> Asp)], is used to determine the relative contributions of Tyr alpha42 and the penultimate tyrosines to the R - T UV resonance Raman difference spectrum. The results both directly support the claim that Tyr alpha42 is the proton acceptor in the T state and expose the potential role of the penultimate tyrosines in coupling the quaternary state to the ligand reactivity.

Dynamics of cyanide binding to ferrous Scapharca inaequivalvis homodimeric hemoglobin.

Flash photolysis experiments have been carried out for the first time on a hemoglobin ferrous cyanide adduct with an 8 ns laser pulse. A 95% nonexponential rebinding process occurs within 2 micros after full photolysis in ferrous cyanide dimeric Scapharca inaequivalvis hemoglobin (HbI), indicating that once photolyzed the cyanide anion is not able to escape from the protein matrix and rebinds to the heme iron. The resonance Raman spectrum of the 10 ns photoproduct is identical to that of the fully relaxed deoxy derivative, indicating that in the ferrous cyanide HbI adduct protein relaxation occurs within 10 ns after photolysis. This behavior is at variance with that of the carbonmonoxy HbI derivative in which very little geminate rebinding is observed and the photoproduct relaxes with a lifetime of 1 micros. The fast relaxation of the cyanide HbI photoproduct can be accounted for by the small perturbation of the heme structure induced by cyanide binding to ferrous HbI. This is consistent with a deoxy-like conformation of the HbI ferrous cyanide adduct and implies that the pathway for relaxation involves only minor local rearrangements of the heme moiety. Photolysis experiments carried out on ferrous cyanide horse myoglobin, which can be saturated only partially, show a qualitatively similar behavior in ligand rebinding, indicating that the geminate process of the cyanide anion is a general phenomenon in hemoproteins.

Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin.

One of the most remarkable structural aspects of Scapharca dimeric hemoglobin is the disruption of a very well-ordered water cluster at the subunit interface upon ligand binding. We have explored the role of these crystallographically observed water molecules by site-directed mutagenesis and osmotic stress techniques. The isosteric mutation of Thr-72-->Val in the interface increases oxygen affinity more than 40-fold with a surprising enhancement of cooperativity. The only significant structural effect of this mutation is to destabilize two ordered water molecules in the deoxy interface. Wild-type Scapharca hemoglobin is strongly sensitive to osmotic conditions. Upon addition of glycerol, striking changes in Raman spectrum of the deoxy form are observed that indicate a transition toward the liganded form. Increased osmotic pressure, which lowers the oxygen affinity in human hemoglobin, raises the oxygen affinity of Scapharca hemoglobin regardless of whether the solute is glycerol, glucose, or sucrose. Analysis of these results provides an estimate of six water molecules lost upon oxygen binding to the dimer, in good agreement with eight predicted from crystal structures. These experiments suggest that the observed cluster of interfacial water molecules plays a crucial role in communication between subunits.

Immune mechanisms in congenital cytomegalovirus infection: activation of CMV-specific T helper cells (CMV-Th) by exogenous IL-2.

Infants with congenital CMV infection have a specific defect in CMV-induced lymphocyte proliferation, providing a model for investigation of mechanisms of viral immune recognition and immune response. In the present study the possible role of a defect in lymphokine activation of CMV-specific T helper cells (Th) was examined. IL-1 activity was detected in supernatants of patient mononuclear cell (MNC) cultures stimulated with CMV. In contrast, no IL-2 activity could be detected in supernatants of CMV-stimulated MNC cultures, whereas PHA induced normal IL-2 production. Addition of low concentrations of either crude TCGF or recombinant IL-2 (rIL-2) resulted in 2-4 fold augmentation of CMV-specific lymphocyte proliferation; exogenous IL-2 had no effect on MNC responses to HSV. CMV-specific Th lines/clones were established from three congenital CMV patients by initial stimulation of MNCs with CMV antigen and 0.1 U/ml rIL-2, followed by repeated stimulation with CMV, HLA-matched allogeneic feeder cells and 10% TCGF. The resulting CMV Th lines/clones proliferated specifically in response to stimulation with CMV antigen and produced endogenous IL-2. Thus, the immune deficiency associated with congenital CMV may either be due to an intrinsic defect in CMV-Th activation or CMV-specific suppressor cell activity.

Reactivity of anti-asialo GM1 serum with tumoricidal and non-tumoricidal mouse macrophages.

All peritoneal macrophage (pM phi) populations studied exhibited some binding of the anti-asGM1 serum as assessed by flow cytometry. The levels of reactivity varied quantitatively among populations, depending on the combination of eliciting and activating agents employed prior to the harvest of pM phi. Resident pM phi contained a very small percentage (4%) of cells that were strongly asGM1+. Any treatment of these cells that induced them to become stimulated or activated increased the percentage of highly asGM1+ cells. Treatments that enhanced anti-asGM1 binding including eliciting pM phi with proteose peptone (16% asGM1+) or Brewer's thioglycollate medium (66% asGM1+), treatment with the activating biological response modifiers (BRMs) MVE-2 (12% asGM1+) and P acnes (18% asGM1+), or treatment with both peptone + MVE-2 (37% asGM1+) or peptone + poly IC/LC (33%). Increased expression of anti-asGM1 was accompanied by some increase in the reactivity of the various pM phi populations to treatment with anti-asGM1 serum. This conclusion was based on the reduced viabilities of cells treated with both an eliciting agent and an activating agent prior to in vitro treatment with anti-asGM1 + C, as well as by reductions in cytolytic activity of pM phi elicited with peptone and activated by MVE-2, following anti-asGM1 treatment in vitro or administration in vivo. Conversely, the cytolytic activity of resident pM phi activated in vivo by MVE-2 or heat-killed P acnes, agents that induced relatively small increases in the percentage of asGM1+ cells, was resistant to the effects of in vivo and/or in vitro treatment with doses of anti-asGM1 serum that inhibit NK activity. These results indicate that stimulation of pM phi by eliciting or activating agents can increase the level of expression of asGM1. This increased expression of asGM1 may be a useful marker for some aspects of macrophage heterogeneity, but increased expression is not necessarily directly related to expression of tumoricidal activity. In fact, the results of this study demonstrate that anti-asGM1 serum can be used for specific depletion of NK activity in vivo in normal mice and in mice treated with at least some BRMs. However, the results also demonstrate that the use of eliciting agents, particularly thioglycollate, or eliciting agents in conjunction with activating agents can cause pM phi to become reactive with anti-asGM1 serum.

Distribution of peritoneal macrophage populations after intravenous injection in mice: differential effects of eliciting and activating agents.

Murine peritoneal macrophages (pM phi) elicited in vivo by intraperitoneal (IP) inoculation of various agents were tested for their homing/distribution patterns after intravenous (IV) adoptive transfer to syngeneic C57BL/6 recipients. Resident pM phi (RpM phi) obtained from normal mice and pM phi elicited by proteose peptone (PpM phi) or thioglycollate broth (TpM phi) exhibited similar homing patterns following IV transfer. After initial arrest in the lungs, these cells rapidly disseminated to liver and spleen, with minimal or no detectable migration to peripheral lymph nodes, intestine, peritoneum, kidney, heart, or retention in the blood. The pattern of results reflected the properties of pM phi themselves, since highly enriched pM phi populations obtained by treatment of crude peritoneal exudate cells with anti-Thy 1.2 + C, or by fractionation on Percoll density gradients, gave similar results. The distribution of pM phi elicited by Brewer's thioglycollate medium (BTpM phi) was markedly different from other pM phi tested. BTpM phi homed rapidly to the lungs and many remained localized there for at least 72 hr with very little migration to the spleen. The distribution of PpM phi could be altered by activation of these cells in vivo through the IP injection of the pyran copolymer, MVE-2, prior to adoptive IV transfer. Activated PpM phi contained a population of highly differentiated, low density pM phi, separable on density gradients, which arrested in the lungs for appreciably longer periods of time than did PpM phi. These cells exhibited reduced ability for migration to the spleen. Macrophage-like (M phi-like) cell lines did not exhibit migration capability, but rather were rapidly cleared from the circulation in a manner similar to other types of tumor cells.

Serum luteinizing hormone levels in Brangus cows following variable suckling intensity and administration of various levels of estrogen.

Fifty Brangus cows were randomly allotted to suckled (S) or nonsuckled (NS) treatment groups on day 20 postpartum. Suckled cows were nursed at 6 hr intervals for 72 hours. Nonsuckled cows were separated from their calves for the entire 72 hours. At 24 hr after initial separation from calves, S and NS cows were given an I.M. challenge of 0, 0.5, 1.0, 2.0 or 4.0 mg estradiol-17beta (E2) to induce a luteinizing hormone (LH) surge (five cows per treatment group). Blood samples were taken at the time of E2 injection and at 2 hr intervals until hr 48 post-injection. Blood serum was analyzed for LH content via radioimmunoassay. Suckled and NS cows manifesting an LH surge after receiving less than 4 mg E2 were 2 of 15 vs 9 of 15 (P<.01), or 4 mg E2 dose were 5 of 5 vs 5 of 5, respectively. Greater serum LH concentrations in NS than S cows were found with dose levels of 0, 0.5 and 1.0 mg E2 (P<.005), but there was no difference by period. Differences by treatment (P<.05) and by period (P<.005) were found at the 2 mg E2 dose. Suckled and NS cows having an LH surge at less than a 4 mg E2 challenge had no differences in LH concentration or timing parameters. Four mg E2 hastened the time of onset of the LH surge (P<.025), time till peak height of the surge (P<.025) and completion of the surge (P<.10). No differences in postpartum interval or conception rate were found between S and nonsuckled. Suckling impairs hypothalamic/pituitary response to low E2 challenge dose and elicits changes in timing parameters in response to high E2 dosage.