Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na₂SO₄ to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.     

Polypeptides encoded by the early region of bacteriophage Mu synthesized in minicells of Escherichia coli

The preferential interaction of calf brain tubulin with glycerol in an aqueous buffer (0.01 m-NaP_i, 0.02 m-NaCl, 10^?4m-GTP, pH 7.0) has been investigated by densimetry. The apparent specific volumes of tubulin at constant chemical potential of the diffusible components were determined at 0, 10, 20 and 30% ($\text{v}\text{v}$) glycerol. Application of multicomponent solution thermodynamics shows that tubulin is preferentially hydrated in aqueous glycerol solvent and that such interaction results in thermodynamic destabilization of the system by raising the chemical potentials of both glycerol and tubulin. Interpreted in terms of the Wyman linkage function, the unfavorable free energy change brought about by the preferential protein-glycerol interaction can account for the glycerol enhancement of tubulin self-assembly in vitro into microtubules as well as offer a rationale for glycerol stabilization of the native tubulin conformation.     

Molecular docking and spectroscopic studies on the interaction of new fifth-generation antibacterial drug ceftobiprole with calf thymus DNA

Abstract

The interaction of the cefobiprole drug with calf thymus DNA (ct-DNA) at physiological pH was investigated by UV-visible spectrophotometry, fluorescence measurement, dynamic viscosity measurements, circular dichroism spectroscopy and molecular modeling. The binding constant obtained of UV–visible was 4?×?10⁴ L mol^?1. Moreover, the results of circular dichroism (CD) and viscosity measurements displayed that the binding of the cefobiprole to ct-DNA can change the conformation of ct-DNA. Furthermore, thermodynamic parameters indicated that hydrogen bond and van der waals play main roles in the binding of cefobiprole to ct-DNA. Optimal results of docking, it can be concluded that ceftobiprole-DNA docked model is in approximate correlation with our experimental results.     

Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State

Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N^∗. Fluorescence resonance energy transfer measurements show that N^∗ then expands rapidly but partially to form an early unfolding intermediate I_E. Fluorescence spectral measurements indicate that both N^∗ and I_E have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, I_L, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when I_L forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.     

The Affinity of DNA-Microtubule Protein Complexes and their Disruption by Tubulin Binding Drugs

Abstract

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar K_app values in the range of 2.1×10⁷ M^?1 to 2.0×10⁸ M^?1. CEN11 DNA-MTP complexes had by far the highest K_app value of 2.0×10⁸ M^?1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites -(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher K_app value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10–100 fold higher drug concentrations to disrupt the CEN 11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383–392,1984; Marx and Denial, Molecular Basis of Cancer 172B,65-15 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.     

Equilibrium unfolding of <Emphasis Type="Italic">A. niger</Emphasis> RNase: pH dependence of chemical and thermal denaturation

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A_MG1) into another (I_MG2).     

A spectral investigation of the copper(II) complex of the antitumor compound bleomycin

A thorough spectral investigation of the copper(II) complex of the antitumor compound, bleomycin, has been carried out in solution employing optical, difference optical, electron spin resonance, and circular dichroism techniques. The optical spectrum of a pH = 7 solution of the 1:1 complex between copper(II) and bleomycin is characterized by a broad weak band in the visible region (λ_max = 610 nm) that cannot be resolved and intense ultraviolet bands at 317 (? = 2800), 327 (shoulder), 250 (? = 4700), and 257 nm (shoulder). The circular dichroism spectrum in the visible region shows the broad and weak visible absorption band contains at least three components (558, 675, and 880 nm) that are likely to be “d-d” in origin. The electron spin resonance spectrum is characteristic of a tetragonal d⁹ copper(II) system showing no rhombic distoritions at X-band frequencies (g_x = g_y ± 0.002). The spin Hamiltonian parameters for the pH = 7.0 solution corrected for second order effects are A_∥ = 177 × 10^?4 cm^?1, A_⊥ ? 15 × 10^?4 cm^?1, g_∥ = 2.214, g_⊥ = 2.039. Most interesting was the observation of extra hyperfine splitting due to endogenous nitrogen coordination in a 30% glycerol glass (A^N = 12.0 × 10^?4 cm^?1). That pattern is best interpreted as a seven-line sequence associated with three liganded nitrogens. A dramatic change in all spectral properties occurs when the pH of the copper(II)-bleomycin complex is lowered to 2.5. All these data taken together suggest a CuN₃O coordination complex in solution. Details and justifications as well as a discussion of the limitations of the interpretations are presented.     

Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State

Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N^∗. Fluorescence resonance energy transfer measurements show that N^∗ then expands rapidly but partially to form an early unfolding intermediate I_E. Fluorescence spectral measurements indicate that both N^∗ and I_E have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, I_L, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when I_L forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.     

Ultraviolet circular dichroism of polyproline and oriented collagen

The ultraviolet absorption, linear dichroism, circular dichroism, and oriented circular dichroism of collagen are reported and the spectra are resolved into a self-consistent set of bands in accord with exciton theory. The parallel band at 200 nm has 40% of the π → π* intensity; the perpendicular band is placed at 189 nm yielding a splitting of 2700 cm^?1. The circular dichroism is resolved into two Gaussians at λ_∥ and λ_τ (rotational strengths +14 × 10^?40 and ?32 × 10^?40 esu². cm²) plus a large non-Gaussian (“helix”) band with ampplitude ?25,000° at 201 nm. These data appear to be in reasonably good accord with recent calculations. Measurements of the absorption, linear dichroism and circular dichroism of polyproline I and II are also reported and are resolved into their component bands. Polyproline I is in good accord with exciton theory, whereas polyproline II remains unsatisfactory.     

Human placental alkaline phosphatase: Effects on conformation by ligands which alter catalytic activity

The conformation of human placental alkaline phosphatase (EC 3.1.3.1) has been studied using the spectroscopic structural probes of pH difference spectroscopy, solvent perturbation difference spectroscopy, and circular dichroism. Of the 37 ± 1 tyrosine residues in placental alkaline phosphatase (PAP), 5 ± 1 residues are observed by pH difference spectroscopy to be “free” and presumed to be located on the surface of the enzyme molecule. The ionization of these 5 “free” tyrosyl groups is not time dependent and is reversible with a pK_app of 10.29. The remaining 32 ± 1 tyrosines are considered “buried” and ionization is observed to be both time dependent and irreversible. Treatment of the enzyme with 4 m guanidine-hydrochloride normalizes all 37 ± 1 tyrosine residues (pK_app = 10.08). The difference pH titration studies thus provide spectrophotometric evidence for a change in molecular conformation of PAP in the pH region of 10.5. Using solvent perturbation difference spectroscopy and circular dichroism, the local environments of tyrosine and tryptophan residues were elucidated for the native enzyme and the enzyme in the presence of ligands that influence catalytic function: inorganic phosphate (competitive inhibitor), l-phenylalanine (uncompetitive inhibitor), d-phenylalanine (noninhibitor). and Mg²⁺ ion (activator). The spectral observations from these studies led to the following interpretations: (i) the binding of inorganic phosphate, a competitive inhibitor, induces a conformational change in the enzyme that may alter the active site and thereby decrease enzyme catalytic function; (ii) perturbation with l-phenylalanine gives spectral results indicating a conformational change consistent with the postulate that this uncompetitive inhibitor prevents the dissociation of the phosphoryl enzyme intermediate; and (iii) Mg²⁺ ion causes a slight separation of the enzyme subunits, which could increase accessibility to the active site and, thus, enzyme activity.     

Confomational and molecular responses to pH variation of the purified membrane adenosine triphospate of Micrococcus lysodeikticus

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95 %. pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 °C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10^?4 M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20–30 % of the activity could be recovered when the pH was returned to 7.5.In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel clectrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.     

Biochemical determination of tubulin-microtubule equilibrium in cultured cells.

The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH₃ cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34–41% of tissue culture cell tubulin. Colchicine (10^?6 to 10^?5m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.     

Nuclear magnetic resonance studies of the unfolding of pancreatic ribonuclease. II. Unfolding by urea and guanidine hydrochloride.

The unfolding of ribonuclease by urea and guanidine hydrochloride has been studied by ¹H nuclear magnetic resonance spectroscopy, under conditions where the unfolding is fully reversible and concentration-independent. Both urea and guanidine produce marked changes in the chemical shift of the histidine C₍₂₎H resonances, together with small changes in other regions of the spectrum, at concentrations (0.1 to 1.0 m) far below those which are required for gross unfolding of the protein. The changes in area of the histidine C₍₂₎H resonances through the major unfolding transition produced by these denaturants give evidence for the existence of at least two intermediates in the unfolding process. The “order of unfolding” of the histidine residues is closely similar for both urea and guanidine hydrochloride unfolding, and also similar to that found for thermal unfolding at low pH (see Benz &; Roberts, 1975) accompanying paper.     

Revisiting absorbance at 230 nm as a protein unfolding probe

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A₂₃₀) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A₂₃₀ as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A₂₃₀. Significant changes in A₂₃₀ were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A₂₃₀ in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A₂₃₀ under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A₂₃₀ allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A₂₃₀ is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.     

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Abstract

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, ¹H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440?nm upon excitation around 380?nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by ¹H NMR study. The binding constants are calculated to be 5.3?×?10⁷ and 6.8?×?10⁶ M^?1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC₅₀ in the order of 0.023?mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.

Communicated by Ramaswamy H. Sarma     

Viscosity and optical activity of chondroitin sulfate C

Chondroitin sulfate C (CSC), isolated from shark vertebral mucoprotein, has a molecular weight of 0.2 × 10⁴–5.5 × 10⁴ in both NaCl and CaCl₂ solutions. Optical rotatory dispersion and circular dichroism of CSC reveal a strong, negative, optically active band near 210 mμ, arising from the carboxylate and N-acetyl groups. Results of similar studies of glucuronic acid and N-acetyl-D -galactosamine suggest that the N-acetyl group contributes more to the rotations of CSC than does the carboxylate group, but the acidification of the carboxylate groups largely accounts for the change in magnitude and position of the circular dichroic bands of CSC at low pH.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

Regulation of catecholamine synthesis in rat brain synaptosomes

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent K_m of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10^?6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10^?7m ; noradrenaline, 25% inhibition at 5 ± 10^?6m ;and serotonin, 30% inhibition at 10^?5m . The catecholamine-induced inhibition of synthesis was antagonized by pre-incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra-synaptosomal amine levels, and that short-term exposure to depolarizing concentrations of potassium can increase synthesis.     

Release of [14C]tyrosine from tubulinyl-[14C]tyrosine by brain extract. Separation of a carboxypeptidase from tubulin-tyrosine ligase

Summary The carboxypeptidase previously described³ that releases tyrosine from tubulinyl-tyrosine was obtained from rat brain preparation free of tubulin-tyrosine ligase. The enzyme was purified 24-fold. Its activity was increased by 2 mm MgCl₂ or 30 mm KCl. Mercaptoethanol (50 mm), colchicine (0.2 mm) and tyrosine (0.2 mm) showed practically no effect on the release of tyrosine whereas iodoacetate (2 mm), deoxycholate (0.5%), CuCl₂ (0.1 mm), ZnC1₂ (0.1 mm) and NaCl or KCI (240 mm) had a strong inhibitory effect. The optimal pH of this enzyme. was 6.3–7.A preparation containing tubulin-tyrosine ligase free of carboxypeptidase was also obtained. This preparation catalyzed the release of tyrosine from tyrosinated tubulin in the presence of ADP, Mg²⁺, K^– and Pi and the incorporation of tyrosine into tubulin. For the releasing activity the optimal concentration of MgCl₂ was 3–20 mm and of KCl was 10–30 mm. For ADP the maximal activity was at 0.3 mm or higher.An important difference between the activities of the carboxypeptidase and the ligase was that the former was active on denatured tubulin whereas the latter was not.     

Magnetic circular dichroism analysis of the ihp effect on spin equilibria in human ferric hemoglobins1

Magnetic circular dichroism (MCD) spectroscopy has been used to explore the connection between optical spectra and the high spin population of several hemoglobins under various conditions. It is found that the effectiveness of IHP in inducing spectral changes can be markedly affected by solvent. For example, the IHP-induced spectral changes in the visible region for nitritomethemoglobin-A in mixed buffer solvent systems (glycerol or polyethylene glycol (PEG), mw 190–210) are more than double those observed in aqueous buffers. We estimate that IHP induces a mix of R/T forms in bis-tris phosphate buffers, for NO₂^?metHb that is only about 50% T form. While PEG and glycerol both lead to enhanced IHP-induced spectral differences, they behave differently in two aspects. PEG shifts the visible MCD and absorption spectra of F^?metHb-A. supposedly already biased towards the T form by ligand, in the same direction that IHP does. PEG also maximizes the spin state changes with IHP for three R form hemoglobins and N₃^?metHb-A, and so appears to stabilize the T form in all cases. Glycerol does not. In addition, the apparent binding constant for NO₂^? to H₂OmetHb-A differs between these two solvents. Comparison of the data from several hemoglobins leads to the conclusion that the changes in spin state distributions induced by IHP correlate well with quarternary structure for a given hemoglobin. An analogous correlation amongst various proteins between initial spin state distribution (IHP) absent) and quarternary structure is not found.