Chemical and physiological interactions between e-liquid constituents: cause for concern?

Studies of Electronic Nicotine Delivery Systems (ENDS) toxicity have largely focused on individual components such as flavour additives, base e-liquid ingredients (propylene glycol, glycerol), device characteristics (eg, model, components, wattage), use behaviour, etc. However, vaping involves inhalation of chemical mixtures and interactions between compounds can occur that can lead to different toxicities than toxicity of the individual components. Methods based on the additive toxicity of individual chemical components to estimate the health risks of complex mixtures can result in the overestimation or underestimation of exposure risks, since interactions between components are under-investigated. In the case of ENDS, the potential of elevated toxicity resulting from chemical reactions and interactions is enhanced due to high operating temperatures and the metallic surface of the heating element. With the recent availability of a wide range of e-liquid constituents and popularity of do-it-yourself creation of e-liquid mixtures, the need to understand chemical and physiological impacts of chemical combinations in ENDS e-liquids and aerosols is immediate. There is a significant current knowledge gap concerning how specific combinations of ENDS chemical ingredients result in synergistic or antagonistic interactions. This commentary aims to review the current understanding of chemical reactions between e-liquid components, interactions between additives, chemical reactions that occur during vaping and aerosol properties and biomolecular interactions, all of which may impact physiological health.

A spectroscopic investigation of the binding interactions between 4,5-dihydroxyxanthone and heme.

In order to investigate one possible mechanism by which xanthones inhibit growth of malaria-causing Plasmodium parasites, optical and NMR spectroscopic studies were performed on a prototypic xanthone, 4,5-dihydroxyxanthone (45X2), upon its complexation to heme. The 45X2 x heme complex stoichiometry in aqueous solution was found to be 1:2; this interaction was non-cooperative, and exhibited a very similar heme complex dissociation constant (K(d)=5.1 x 10(-6)) as observed for the common antimalarial agents, chloroquine and quinine. The 45X2 x heme(2) complex formation was found to be both pH- and solvent-dependent, with clear evidence of the xanthone carbonyl moiety coordinating with the iron of heme. Hydrogen bonding between the hydroxyl groups of 45X2 and the propionate side chains of heme, as well as pi-pi stacking between both aromatic systems appeared to contribute to the overall stability of the 45X2 x heme(2) complex, as judged by 1H NMR. It was concluded that 45X2 forms a complex with a heme dimer in aqueous solution, and that this interaction can be generalized to account for its in vivo detrimental effect of parasite growth through an effective inhibition of hemozoin aggregate formation.

Sweet is stable: glycosylation stabilizes collagen.

For most collagens, the melting temperature (T(m)) of the triple-helical structure of collagen correlates with the total content of proline (Pro) and 4-trans-hydroxyproline (Hyp) in the Xaa and Yaa positions of the -Gly-Xaa-Yaa- triplet repeat. The cuticle collagen of the deep-sea hydrothermal vent worm Riftia pachyptila, despite a very low content of Pro and Hyp, has a relatively high thermal stability. Rather than Hyp occupying the Yaa position, as is normally found in mammalian collagens, this position is occupied by threonine (Thr) which is O-glycosylated. We compare the triple-helix forming propensities in water of two model peptides, Ac-(Gly-Pro-Thr)(10)-NH(2) and Ac-(Gly-Pro-Thr(Galbeta))(10)-NH(2), and show that a collagen triple-helix structure is only achieved after glycosylation of Thr. Thus, we show for the first time that glycosylation is required for the formation of a stable tertiary structure and that this modification represents an alternative way of stabilizing the collagen triple-helix that is independent of the presence of Hyp.

Solution structures of human immunodeficiency virus type 1 (HIV-1) and moloney murine leukemia virus (MoMLV) capsid protein major-homology-region peptide analogs by NMR spectroscopy.

The capsid domain of retroviral Gag proteins possesses a single highly conserved subdomain termed the major homology region (MHR). While the mutagenesis of residues in the MHR will impair virus infectivity, the precise solution structure and function of the MHR is not known. To aid the structure/function characterization of the MHR, the structures of synthetic peptides encompassing the MHR of the human immunodeficiency virus type I (HIV-1) and Moloney murine leukemia virus (MoMLV) capsid proteins were investigated by several techniques. Homology-based secondary-structure prediction suggested that the HIV-1 and MoMLV peptides could form 50% and 38% alpha-helix, respectively. CD studies indicated that, in the presence of 50% trifluoroethanol, the HIV-1 peptide adopts an alpha-helical structure over half of its length, while the MoMLV peptide is over one third alpha-helix. Further analysis by 1H-NMR suggested that the C-terminal portion of the MHR of each virus forms a helix in aqueous solution. Distance-geometry structures of each peptide were calculated from NOE distance restraints and were refined by restrained molecular dynamics. The C-terminal halves of both peptides were observed to be in an alpha-helical conformation, while the N-terminal halves were disordered. Furthermore, both helices were amphipathic with high conservation of amino acid side-chain character, suggesting that a conserved helical MHR C-terminus is essential to retroviral capsid protein function.

Proton resonance assignments and ligand exchange kinetics in high-spin and mixed-spin myoglobin complexes using two-dimensional exchange spectroscopy.

The task of assigning resonances in proton nuclear magnetic resonance spectra of paramagnetic heme proteins can be an arduous process, but with the development of multi-dimensional NMR methods the situation has improved. It is demonstrated here that two-dimensional exchange spectroscopic experiments can be used to obtain to assignment correlations for the heme protons of methydroxy-, metthiocyano-, metaquo-, and metimidazole-myoglobin forms. All the assignments are unambiguous and straightforward when the temperature and mixing times are adjusted to minimize nuclear Overhauser cross-peaks from each complex. Moreover, saturation transfer experiments allow the study of ligand binding kinetics. The exchange rates between metaquo- and metimidazole- (or methyl substituted imidazole) myoglobin complexes are estimated. The differences between the exchange rates reflect differences in the hydrophobic and steric interactions between the ligands and the protein moiety.

Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten.

The structural basis of the binding of phenylphosphocholine haptens to antibodies was studied. This was done by preparing antibodies and testing binding to conjugates of phenylphosphocholine. The choice of haptens was made in order to evaluate the contribution of the carrier to binding, and its effect on hapten conformation in the active site. Thus, phosphocholine (PC) was diazophenyl-linked to tyrosine or histidine as single amino acid carriers and to tripeptides or octapeptides containing tyrosine or histidine as central amino acids to which PC was attached. Relative affinity was assessed by inhibition enzyme-linked immunosorbent assay (ELISA) and binding constants were determined by fluorescence quenching. Fluorinated haptens were used to determine the kinetics of binding using 19F nuclear magnetic resonance. The transferred nuclear Overhauser effect was used to characterize conformation of the bound hapten. We had previously shown that nitrophenylphosphocholine unlinked to carrier is bound in the active site as a bent structure [Bruderer, U., Peyton, D. H., Barbar, E., Fellman, J. H., & Rittenberg, M. B. (1992) Biochemistry 31, 584-589]. We show here that this same bent conformation is retained in the active site regardless of the neighboring carrier or the conformation of the hapten in the unbound conjugate. The presence of the carrier residues in the bound state does, however, influence affinity.

Spectroscopic study of an HIV-1 capsid protein major homology region peptide analog.

The capsid (CA) domain of retroviral Gag proteins possesses one subdomain, the major homology region (MHR), which is conserved among nearly all avian and mammalian retroviruses. While it is known that the mutagenesis of residues in the MHR will impair virus infectivity, the precise structure and function of the MHR is not known. In order to obtain further information on the MHR, we have examined the structure of a synthetic peptide encompassing the MHR of human immunodeficiency virus type I (HIV-1) CA protein. Multiple sequence alignment and secondary structure prediction indicate that the peptide could form 50% alpha-helix and 10% beta-sheet. In addition, circular dichroism studies indicate that, in the presence of 50% trifluoroethanol (TFE), the peptide adopts an alpha-helical structure over half of its length. Further analysis by proton nuclear magnetic resonance spectroscopy suggests that the C-terminal portion of the MHR forms a helix in aqueous solution. Upon the addition of TFE, the position of the helix remains nearly constant, but the magnitude of the changes in H alpha chemical shifts of the residues indicate a more stable helix. These results suggest that a helical C-terminus of retroviral MHRs may be integral to the function of this region.

Variable-temperature study of the heme-reorientation process in equine myoglobin.

The redistribution of the initially-formed myoglobin heme-insertion isomers from the initially formed 50/50 mixture to the equilibrium ratio of 90/10 has long been assumed to occur by one of two mechanisms, both of which require the rupture of the heme iron-protein bond (La Mar, G.N., Toi, H. and Krishnamoorthi, K. (1984) J. Am. Chem. Soc. 106, 6395-6401). In this study we compared the use of nuclear magnetic resonance and optical spectroscopic techniques as methods for studying the reorientation of heme within myoglobin. We found that kinetics determinations of the heme insertion isomer redistribution process in Mb by optical spectroscopy are quantitatively compatible with the results obtained by nuclear magnetic resonance spectroscopy. A variable-temperature analysis for horse myoglobin using the optical method at pH 8.4 +/- 0.1 yielded the following activation energy parameters: delta H++ = 31 kcal/mol, delta S++ = 34 cal/mol per K, and delta G++21 degrees C = 21 kcal/mol. The value of delta G++ expected for complete dissociation of the heme from myoglobin can be estimated, from its dissociation constant and insertion rate, to be on the order of 23-27 kcal/mol under the same conditions as our determination. Therefore, although the mechanism for heme reorientation in Mb is likely non-dissociative, it has an activation energy which is not far from the lower bound expected for a complete-dissociation/recombination mechanism. Our measured entropy of activation is not especially large, perhaps owing to a large contribution by the solvent.

NMR behavior of the aromatic protons of bovine neurophysin-I and its peptide complexes: implications for solution structure and for function.

The NMR behavior of the aromatic protons of bovine neurophysin-I and its complexes was interpreted with reference to the 2.8 A crystal structure of the dipeptide complex of bovine neurophysin-II and to mechanisms underlying the thermodynamic linkage between neurophysin dimerization and peptide binding. Large binding-induced shifts in the ring proton signals of Tyr-2 of ligand peptides (approximately 0.5 ppm upfield and approximately 0.35 ppm downfield at 25 degrees C for the 3,5- and 2,6-ring protons, respectively) were demonstrated. Consistent with the crystal structure, and in disagreement with conclusions by other investigators, evidence is presented indicating the absence of dipolar contact between Tyr-2 ring protons and protein Phe ring protons. The large binding-induced shifts are attributed to a strong influence of proximal neurophysin carbonyl and disulfide groups on the bound Tyr-2 ring, of potential importance in binding specificity. Resolution of the behavior of neurophysin Phe residues -22 and -35 and of their proton NOE contacts provided insights into the conformational changes associated with peptide binding and with dimerization. Within the amino domain of the protein, as evidenced by the behavior of interface residue Phe-35 and its NOE contacts, binding-induced changes in the subunit interface appeared to involve principally the junction between this interface region and the 3,10-helix that connects it to the binding site in the liganded state. By contrast, as judged by the NOE contacts of His-80, the corresponding interface participant of the carboxyl domain, peptide binding induced a marked decrease in side-chain mobility within the carboxyl domain segment of the interface. Interactions of Phe-22 with protons assigned to Ala-68, neither of which is an interface participant, were demonstrated to be markedly altered both by dimerization in the unliganded state and by peptide binding to the dimer. Since Phe-22 is adjacent to the peptide-binding site, the results collectively support a model in which conformational differences between unliganded monomer and dimer are important contributors to the preferential binding of peptide to the dimer and indicate that the amino and carboxyl domain segments of the interface, which are homologous, are affected differently by peptide binding.

Isolation and identification of two potent neurotoxins, aspartic acid and glutamic acid, from yellow star thistle (Centaurea solstitialis).

Horses grazing for prolonged periods on yellow star thistle (YST), a plant which grows wild in western parts of the United States, develop an extrapyramidal disorder known as nigropallidal encephalomalacia (NPE). Attempts have been made to identify, isolate, and characterize the toxins responsible for the disease in animals. Using the organotypic tissue culture system on mouse cortical explants as a specific assay method for neurotoxicological evaluation, it has been possible to isolate and characterize two potent neuroexcitotoxic compounds, aspartic and glutamic acids, the former being the major toxic component in the alcoholic extract of the plant. There is also evidence that other neurotoxic compounds are present in the extract. The detailed procedure for isolation and characterization of these compounds is given here.

Roles of heme iron-coordinating histidine residues of human hemopexin expressed in baculovirus-infected insect cells.

Hemopexin (Hx), the major heme-binding plasma glycoprotein, scavenges circulating heme and performs an antioxidant function. In the present study, human Hx was expressed in a baculovirus system and its presumed essential His residues were mutated to Thr as a means of investigating their participation in heme binding. The recombinant Hx proteins were purified by sequential chromatography on Con A-agarose and SP-Sepharose. The purified recombinant wild-type Hx retained its heme binding. The binding constant for heme was considerably reduced, however, suggesting that glycosylation contributes critically to the heme binding property of Hx. Mutation either at His-127 or at His-56 plus His-127, but not at His-56 per se, reduced the affinity for heme by an order of magnitude relative to wild-type Hx. It is concluded that His-127 contributes to the high affinity for heme. We recorded proton NMR spectra to investigate the possibility that the degree of high-spin content is increased by deletion of an axial His-iron coordination. 1H NMR data indicate that each of the single-mutant heme-Hx complexes is predominantly low-spin, perhaps owing to coordination of the heme iron by the Thr side-chain oxygen or water oxygen coordinating to the iron.

Proton NMR study of the heme complex of hemopexin.

Proton nuclear magnetic resonance spectroscopy of the complex of heme with hemopexin, a plasma protein with an exceptionally high affinity for heme, is reported. Characteristic spectra are shown for heme.hemopexin of cow, human, rabbit, and rat. Each of these spectra demonstrate that the iron of heme bound by hemopexin is paramagnetic and low-spin. Rabbit heme.hemopexin, which exhibits the best signal-to-noise ratio, is studied in detail. Deuterium isotope labeling experiments indicate that the methyls in heme positions 1-, 3-, and 8- are resolved downfield from the protein envelope of resonances; the 5-methyl may lie in the -5 to +12 ppm region. Two-dimensional nuclear Overhauser effect spectroscopy locates other protons of the heme periphery, including from the 2-vinyl. Strongly relaxed upfield resonances are identified and assigned to protons on the axial ligands. Cyanide interaction with heme.hemopexin produces an additional low-spin adduct.

AHA- heterodimer of a class-2 uncoupler: pentachlorophenol.

AHA- heterodimers formed by association of neutral molecules of weak acid (HA) with its conjugate anion (A-) have been proposed to be the charged membrane-permeable species of class-2 uncouplers. Past attempts to extract and identify AHA- heterodimers failed. We have measured optical spectra of HA+A- (1:1) solutions of pentachlorophenol (PCP) in various solvents and in the presence of PC liposomes. Optical studies were supplemented by nuclear magnetic resonance measurements of HA+A- (1:1) solutions of PCP in dichloroethane to gain insight into the formation of AHA- species in lipid membranes. From these experiments, we found evidence for AHA- formation in non-hydrogen-bonding solvents, then reported the AHA- formation constant Kf and the molar absorptivity epsilon AHA-(lambda). Kf decreases with increasing dielectric constant, kappa, from 1210 +/- 130 M-1 for dichloroethane (kappa 10.7), to 340 +/- 34 M-1 for acetonitrile (kappa 37.5); Kf also decreases with increasing concentration of water. In hydrogen-bonding solvents, octanol (kappa 10.3) and methanol (kappa 33.5) and in liposomes, AHA- heterodimers are not observed optically. We estimate Kf for PCP in lipid bilayers from a combination of data on membrane electrical conductivity and surface density of adsorbed PCP. Our estimate for lipid bilayer, 0.005 < Kf < 0.5 M-1, is consistent with our inability to detect the AHA- species optically in liposome suspensions. We propose that penetration of water into the membrane inhibits formation of AHA- in lipid bilayers.

Proton NMR study of the interaction of tin(IV) protoporphyrin IX monomers and dimers with apomyoglobin.

Events during the reconstitution of apomyoglobin to form the holoprotein were probed by porphyrin-metal substitution. Thus interactions between tin(IV) protoporphyrin IX (SnPP) and equine apomyoglobin (apoEqMb), and between tin(IV) protoporphyrin IX dimers [(SnPP)2] and apoEqMb, were observed by 1H NMR and optical absorbance spectroscopic techniques. The chief advantages of using SnPP are that products and intermediates can easily be related to SnPP.EqMb which has been studied [Deeb, R.S., & Peyton, D.H. (1991) J. Biol. Chem. 266, 3728-3733] and that at least one step during reconstitution is slowed considerably as compared to heme. Reactions of apoEqMb with SnPP and (SnPP)2 produce different intermediates, although the final product, SnPP.EqMb, is the same for each. An intermediate observed for reaction of SnPP with apoEqMb at pH 10 is in exchange with free SnPP, with the observed rate constant koff approximately 1 s-1. meso-Proton resonances were assigned for this intermediate by correlation to SnPP resonances via chemical exchange. The intermediate observed for reaction of (SnPP)2 with apoEqMb at pH 7.5 is heterogeneous. The reaction of either SnPP or (SnPP)2 with apoEqMb at neutral pH produces another species which may be the alternate porphyrin-insertion isomer arising from a 180 degree rotation about the alpha, gamma-meso axis of the porphyrin. Although optical absorbance spectroscopy of the Soret region shows evidence for each reaction, only in combination with 1H NMR are the various processes assigned.

Hapten conformation in the combining site of antibodies that bind phenylphosphocholine.

We have shown previously that anti-phenylphosphocholine antibodies elicited by phosphocholine-keyhole limpet hemocyanin can be divided into two populations according to their ability to recognize the two hapten analogues p-nitrophenylphosphocholine (NPPC) and p-nitrophenyl 3,3-dimethylbutyl phosphate (NPDBP). These analogues differ from each other in that NPPC has a positively charged nitrogen in the choline moiety, whereas NPDBP lacks the positively charged nitrogen. Group II-A antibodies bind only NPPC, whereas group II-B antibodies bind both ligands. Here, by infrared and nuclear magnetic resonance spectroscopic investigations, we find that when free in solution NPPC has a predominantly fixed structure in which the termini approach each other, probably due to electrostatic interactions within the molecule; this "bent" structural feature is retained when the ligand is bound by antibody. In contrast, the structure of unbound NPDBP is less fixed, being characterized by rapidly interchanging conformations corresponding to an open chain structure with less overall proximity of the termini compared to NPPC. The overall shape of NPPC is essentially unaltered by binding, whereas in the case of NPDBP what was a minor conformation in the unbound state becomes the predominate conformation of the bound ligand. Thus, our results are consistent with these antibodies providing a molecular template for stabilizing the conformation of the bound ligand.

Proton nuclear magnetic resonance study of the solution distal histidine orientation in monomeric Chironomus thummi thummi cyanomet hemoglobins. Dynamic stability of the heme pocket as monitored by labile proton exchange.

The 1H nuclear magnetic resonance spectral characteristics of the cyano-Met form of Chironomus thummi thummi monomeric hemoglobins I, III and IV in 1H2O solvent are reported. A set of four exchangeable hyperfine-shifted resonances is found for each of the two heme-insertion isomers in the hyperfine-shifted region downfield of ten parts per million. An analysis of relaxation, exchange rates and nuclear Overhauser effects leads to assignments for all these resonances to histidine F8 and the side-chains of histidine E7 and arginine FG3. It is evident that in aqueous solution, the side-chain from histidine E7 does not occupy two orientations, as found for the solid state, rather the histidine E7 side-chain adopts a conformation similar to that of sperm whale myoglobin or hemoglobin A, oriented into the heme pocket and in contact with the bound ligand. Evidence is presented to show that the allosteric transition in the Chironomus thummi thummi hemoglobins arises from the "trans effect". An analysis of the exchange with bulk solvent of the assigned histidine E7 labile proton confirms that the group is completely buried within the heme pocket in a manner similar to that found for sperm whale cyano-Met myoglobin, and that the transient exposure to solvent is no more likely than in mammalian myoglobins with the "normal" distal histidine orientation. Finally, a comparison of solvent access to the heme pocket of the three monomeric C. thummi thummi hemoglobins, as measured from proton exchange rates of heme pocket protons, is made and correlated to binding studies with the diffusible small molecules such as O2.

Proton NMR investigation of the reconstitution of equine myoglobin with hemin dicyanide. Evidence for late formation of the proximal His93F8-iron bond.

Reconstitution of apoequine myoglobin (apoEqMb) with hemin dicyanide (FePPIX(CN)2) was monitored by 1H NMR spectroscopy to gain information about the sequence of events leading to metEqMbCN. At least one step in the pathway is slow enough to allow us to follow the time-dependence of formation of the product, a mixture of heme-insertion isomers characterized by others (Jue, T., Krishnamoorthi, R. and La Mar, G.N. (1983) J. Am. Chem. Soc. 105, 5701-5703; Lecomte, J.T.J., Johnson, R.D. and La Mar, G.N. (1985) Biochim. Biophys. Acta 829, 268-274). However, in contrast to all previously reported Mb-FePPIX reconstitutions, we find that the initial ratio of heme-insertion isomers is not 1:1. This mixture is, instead, found to be enhanced in the isomer which dominates at equilibrium. This is taken as evidence for a '[FePPIX(CN)2.EqMb]' intermediate which lacks the proximal His93F8-Fe bond and which proceeds quickly toward an equilibrium ratio of heme-insertion isomers. Therefore the heme-insertion isomer ratio is frozen only when the proximal His93F8-Fe bond is formed. The difference in this ratio of heme-insertion isomers between EqMb (4.5:1) and SwMb (2.5:1) likely reflects the amino acid substitution: Lys----Arg45CD3 (EqMb----SwMb).

1H NMR resonance assignments in a paramagnetic heme protein by two-dimensional spectroscopy: heme resonances in equine met-azido myoglobin.

Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional 1H NMR experiments NOESY and COSY. Metazido myoglobin provides a useful test case for the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as models for the physiological forms of hemoglobin. Third, a few sperm whale met-azido myoglobin resonances have been previously assigned, which permits a comparison of assignments for these similar proteins, and a check of the method presented here.

1H and 119Sn magnetic resonance study of the SnIV protoporphyrin IX complex of equine myoglobin. Structure of the porphyrin-binding pocket.

Tin protoporphyrin IX (SnPP) is being used in the treatment of hyperbilirubinemia. We have studied the SnPP complex with equine myoglobin (EqMb) by 1H and 119Sn nuclear magnetic resonance spectroscopy (NMR) as a general model for SnPP interaction with hemoproteins. The complex formed from SnPP and EqMb, SnPP.EqMb, was found to have essentially the same porphyrin-binding pocket as EqMbCO, including the same porphyrin orientation in the major form of EqMbCO. 119Sn NMR spectroscopy has been used to demonstrate that the proximal His93F8-metal coordination is likely to be intact in SnPP.EqMb. Minor shifts in the side chain positions of some of the residues are indicated, possibly reflecting the presence of water in the sixth coordination site. SnPP.EqMb appears to be stable; it persists at room temperature for weeks and exhibits very slow exchange rates (2H for 1H) for a large number of amide protons in the pH range 7-9.

Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin.

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F., & Brunori, M. (1987) Biochem. J. 246, 787-789].