Phascolopsis Gouldii Research Articles

EPR investigation of libration motion of spin labeled hemerythrin

Reported here are room-temperature continuous wave X-band Electron Paramagnetic Resonance (EPR) spectra of the non-heme di-iron protein hemerythrin (Hr), spin labeled at position 51C in different viscous media, illustrating the mobility and oligomeric recombination tendency of the Phascolopsis gouldii Hr. The mobility of a spin labeled Hr depends on the local viscosity and its connectivity to the nature of the molecular environment (glycerol, PEG4000 and BSA). This provides the basis for a tool useful in directly monitoring Hr in ex vivo samples upon injection within the bloodstream of test animals, for blood substitute research.

Testing rates of planktonic versus benthic predation in the field

Predation is a major source of mortality for the eggs, embryos, and larvae of marine invertebrates. Many studies have measured rates of predation on the developmental stages of marine invertebrates in the lab, but few studies have estimated predation rates in the field. Field studies of predation on developmental stages have generally been limited to organisms in a single environment, with few comparisons of equivalent prey items between habitats. These limitations have prevented comparisons of the relative risks of development in planktonic and benthic habitats. To determine the relative risks of predation for free-living eggs, embryos, and larvae, we measured loss rates for agarose baits flavored with the eggs of two species of marine invertebrates on separate coasts of the United States. First, we deployed agarose baits flavored with eggs of the sand dollar, Dendraster excentricus (Eschscholtz), in planktonic and benthic habitats in Parks Bay, Shaw Island, Washington. We subsequently deployed agarose baits flavored with eggs of the sipunculan Phascolopsis gouldii (Fisher), in planktonic and benthic habitats in Ewin Narrows, Harpswell, Maine. In addition, we measured loss rates of live, tethered megalopae of the Dungeness crab, Cancer magister (Dana) in Washington. For both agarose baits and tethered megalopae, loss rates were highest in benthic habitats. Loss rates of agarose baits flavored with sand dollar eggs were nearly two times greater on the benthos than in the plankton. Loss rates of agarose baits flavored with sipunculan eggs were 13 times greater on the benthos than in the plankton. Loss rates of tethered megalopae were 12 times greater on the benthos than in the plankton during the day, and were 28 times greater for benthic megalopae than for planktonic megalopae at night. These results support a basic, yet previously untested assumption of life-history theory, that rates of predation on developmental stages of marine invertebrates are greater on the benthos than in the plankton.

Molecular Evolution and Phylogeny of Sipunculan Hemerythrins

We sequenced seven new hemerythrin (Hr) and myohemerythrin (myoHr) cDNAs from Sipunculus nudus and Golfingia vulgaris vulgaris, thus providing new comparative data that significantly increase the set of the known Hr and myoHr sequences. Bayesian inference, maximum likelihood, and maximum parsimony phylogenetic analyses were performed to investigate the evolutionary relationships among the sipunculan and annelid Hr and myoHr sequences. Annelid myoHrs and sipunculan Hrs were resolved as monophyletic groups. Conversely sipunculan myoHrs did not form a clade. The Hrs having an octameric quaternary structure were resolved as a monophyletic group. The octameric cluster includes the Hr sequences of G. v. vulgaris, Themiste zostericola, Themiste discriptum, and Phascolopsis gouldii. Siphonosoma cumanense Hr, which has a trimeric quaternary structure, assumes a sister group position of the octameric clade. The S. nudus Hrs, having a quaternary structure that is not well resolved, assume an isolate position within the Hrs clade. Likelihood-based analyses reveal that purifying selection mainly characterized the evolution of Hr and myoHr. We suggest that starting from a common gene ancestor, two distinct quaternary structures evolved in the sipunculan Hrs and this differentiation was probably favored by the acquisition of distinct physiological advantages.

The O(2) binding pocket of myohemerythrin: role of a conserved leucine.

A conserved O(2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), namely, L104, was mutated to several other residues, and the effects on O(2) association and dissociation rates, O(2) affinity, and autoxidation were examined. The L104V, -F, and -Y myoHrs formed stable O(2) adducts whose UV-vis and resonance Raman spectra closely matched those of wild-type oxymyoHr. The L104V mutation produced only minimal effects on either O(2) association or dissociation, whereas the L104F and -Y mutations resulted in 100-300-fold decreases in both O(2) association and dissociation rates. These decreases are attributed to introduction of steric restrictions into the O(2) binding pocket, which are not present in either wild-type or L104V myoHrs. The failure to observe increased O(2) association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not sterically "gate" O(2) entry into or exit from the binding pocket in the rate-determining step(s). L104V myoHr autoxidized approximately 3 times faster than did wild type, whereas L104T autoxidized >10(6) times faster than did wild type. The latter large increase is attributed to increased side chain polarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that restricts water entry into the oxymyoHr binding pocket. Thus, a leucine at position 104 in myoHr appears to have the optimal combination of size and hydrophobicity to facilitate O(2) binding while simultaneously inhibiting autoxidation.

A Leucine Residue “Gates” Solvent but Not O2Access to the Binding Pocket of Phascolopsis gouldiiHemerythrin

A leucine residue, Leu-98, lines the O(2)-binding pocket in all known hemerythrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes (Ala, Val), polarities (Thr, Asp, Asn), and aromaticities (Phe, Tyr, Trp). UV-visible and resonance Raman spectra showed that the di-iron sites in these L98X Hrs are very similar to those in the wild type protein, and several of the L98X hemerythrins formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98X Hrs except for L98W, had second order O(2) association rate constants within a factor of 3 of the wild type value. Similarly, the O(2) dissociation rate constant was essentially unaffected by substitutions of larger (Phe) or smaller (Val, Thr) residues for Leu-98. L98Y Hr showed a 170-fold decrease in the O(2) dissociation rate constant and a large D(2)O effect on this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O(2). Significant increases in autoxidation rates were observed for all of the L98X Hrs other than X = Tyr. These increases in autoxidation rates are attributed to increased solvent access to the binding pocket caused by inefficient packing (Phe), smaller size (Val, Ala), or increased polarity (Thr, Asp, Asn) of the residue 98 side chain. A leucine at position 98 appears to have the optimal size, shape, and hydrophobicity for inhibition of solvent access. Thus, "gating" of small molecule access to the binding pocket of Hr by Leu-98 is not evident for O(2), but is evident for solvent.

Electrospray mass spectrometry studies of non-heme iron-containing proteins.

The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer. The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli. ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms. Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms. Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr). ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein. Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer. The masses of the monomer ions produced in this manner are assigned to the aposubunit, [subunit + Fe - 3H]+, and [subunit + 2Fe - 6 H]+. Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit. Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer. Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3). In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da. Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.

Myohemerythrin from the sipunculid, Phascolopsis gouldii: purification, properties and amino acid sequence

Two previously unknown isoforms, labelled iso I and iso II, of the oxygen-carrying protein, myohemerythrin, have been isolated from carcasses of the sipunculid worm, Phascolopsis gouldii. The two isoforms have non-identical N-terminal amino acid sequences and slightly different absorption spectra in the met form. Far-ultraviolet circular dichroism shows that iso I contains approximately 69% alpha-helix. The complete amino acid sequence for iso I was obtained. The molecular weight calculated from this amino acid sequence and including the active site Fe-O-Fe unit, is 13,829. All of the physical and chemical properties of iso I noted above, including the amino acid sequence, are very similar to those of T. zostericola myohemerythrin. Except for the amino acid sequence, these properties are also very similar to that of a subunit in hemerythrin, the octameric analog found in hemerythrocytes. Only 58 of the 113 residues in P. gouldii hemerythrin are conserved in iso I. Sequence comparisons were used to help identify residues responsible for maintaining the common tertiary and diiron site structures in hemerythrin and myohemerythrin. The seven iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin are conserved in iso I. However, none of the ten residue pairs previously identified as engaging in direct salt-bridge or hydrogen bond interactions between subunits in the hemerythrin octamer are conserved in iso I.

Purification and properties of a membrane-bound NADH-cytochrome- b5 reductase from erythrocytes of the sipunculid worm, Phascolopsis gouldii

The purification to homogeneity of the membrane-bound NADH-cytochrome- b 5 reductase from erythrocytes of the sipunculid, Phascolopsis gouldii is reported. This highly purified reductase has allowed more detailed characterizations of its molecular and kinetic properties than was possible in a previous study (Utecht, R.E. and Kurtz, D.M., Jr. (1988) Biochim. Biophys. Acta 953, 164–178). The reductase has a molecular weight of 34 000 and contains FAD as the prosthetic group. In aqueous solution containing 0.5 vol% Triton X-100, the reductase forms an aggregate of M r ∼ 220 000. A higher purity preparation of P. gouldii erythrocyte cytochrome b 5 was also obtained. The combination of purified, solubilized reductase and cytochrome b 5 was shown to catalyze the quantitative two-electron reduction of [Fe(III),Fe(III)]methemerythrin to [Fe(II),Fe(II)]deoxyhemerythrin by NADH. The P. gouldii NADH-cytochrome- b 5 reductase is the first from hemerythri–containing erythrocytes to be purified and characterized. This methemerythrin reduction system appears to be analogous to methemoglobin reductases from vertebrate erythrocytes.

31P NMR probes of sipunculan erythrocytes containing the O2-carrying protein hemerythrin.

Reported are the first examinations by 31P NMR of erythrocytes containing the non-heme iron O2-carrying protein hemerythrin (Hr). Intact coelomic erythrocytes from the sipunculids Phascolopsis gouldii and Themiste zostericola were shown by 31P NMR to contain O-phosphorylethanolamine and 2-aminoethylphosphonate as the major soluble phosphorus metabolites. This combination of major metabolites appears to be unique to sipunculan erythrocytes. Nucleoside triphosphates and mannose 1-phosphate were present in lower concentrations. The concentration of O-phosphorylethanolamine within P. gouldii erythrocytes was established to be greater than 20 mM. T. zostericola erythrocytes contained relatively high levels of 2-aminoethylphosphonate (on the order of 0.1 M) and lower levels of O-phosphorylethanolamine compared with those of P. gouldii. For P. gouldii and T. zostericola the intracellular pHs were determined to be 7.2 +/- 0.1 and 7.1 +/- 0.1, respectively, in air-equilibrated erythrocytes, and 6.5 +/- 0.1 in anaerobic P. gouldii erythrocytes. O-Phosphorylethanolamine was found to bind weakly to P. gouldii metHr (Kf approximately 7 M-1). This interaction is best characterized by either negative cooperativity or nonspecific binding. O-Phosphorylethanolamine strongly inhibits azide binding to the iron site of P. gouldii metHr at pH 7.2. The rate of azide binding decreases by approximately 85-fold in the presence of 0.33 M O-phosphorylethanolamine. However, neither O-phosphorylethanolamine nor 2-aminoethylphosphonate at 0.33 M was found to have any significant effect on O2 affinity of P. gouldii deoxyHr. Alternative functions for the two metabolites are suggested.

Cytochrome b5 and NADH-cytochrome- b5 reductase from sipunculan erythrocytes; a methemerythrin reduction system from Phascolopsis gouldii

We report the purification and characterization of a soluble cytochrome b 5 from coelomic erythrocytes of the sipunculan worm, Phascolopsis gouldii. We also report the isolation and purification of a membrane-bound NADH-cytochrome- b 5 reductase from these erythrocytes. The non-heme iron protein, hemerythrin (Hr), is known to be the oxygen carrier in these erythrocytes. The aforementioned purified cytochrome b 5 and reductase together catalyze the reduction of P. gouldii [Fe(III),Fe(III)]metHr to [Fe(II),Fe(II)deoxyHr by NADH. EPR spectroscopy demonstrates that a redox process involving formation of the intermediate [Fe(II),Fe(III)]semi-metHr occurs within intact sipunculan erythrocytes as well as in the system of purified components. The rhombic g-tensor of the EPR signal in both cases resembles that of (semi-met) RHr, the form obtained by one-electron reduction of metHr. These observations suggest that cytochrome b 5 and NADH-cytochrome- b 5 reductase in sipunculan erythrocytes function to counteract autoxidation of oxyHr. The sequence of electron flow in the system of purified components is: NADH → NADH-cytochrome- b 5 reductase → cytochrome b 5 → metHr. At pH 7.5, the reduction of metHr in this system occurs in two phases, only the first of which is dependent on concentration of cytochrome b 5. From an analysis of the kinetics and the EPR time-course, we propose that the two phases represent sequential reduction of met- to semi-metHr and reduction of semi-metHr to deoxyHr. This report represents the first demonstration of a physiological system for reduction of metHr.

Allosteric interactions in sipunculid and brachiopod hemerythrins.

Chemical and spectroscopic consequences of allosteric interactions for ligand binding to sipunculid (Phascolopsis gouldii) and brachiopod (Lingula reevii) hemerythrins (Hrs) have been investigated. Possible allosteric effectors for homotropic effects in sipunculid Hrs have been examined, but only reduction in ligand affinity is observed without cooperativity. In contrast to sipunculid Hr, L. reevii Hr binds O2 cooperatively in the pH range 7-8 and exhibits a Bohr effect. Spectroscopic comparisons of the sipunculid and brachiopod Hrs show no significant differences in the active site structures; therefore, modulation of oxygen affinity is attributable to effects linking the site to quaternary structural changes in the octamer. Oxygen equilibria can be fit with a conformational model incorporating a minimum of three states, tensed (T), relaxed (R), and an R-T hybrid. Resonance Raman spectra of L. reevii oxyHr show a shift in the peroxo stretching frequency when the pH is lowered from pH 7.7 (predominantly R oxyHr) to pH 6.3 (a mixture of R, T, and R-T hybrid), but P. gouldii Hr does not have a frequency shift under the same conditions. In contrast to hemoglobins, ligand binding to the deoxy and met forms is noncooperative for brachiopod (and sipunculid) Hrs. It is thus suggested that conformational changes in the protein are linked to the oxidation state change that accompanies oxygenation of the coupled binuclear iron site (deoxy [FeIIFeII]----oxy [FeIIIFeIII]). The total allosteric energy expended in oxygenation is about 1.4 kcal/mol, and such a shift is possible in the relaxed-tense conversion with relatively limited constraints of the iron coordination environment via the protein quaternary structure. The mechanism of cooperativity in the binuclear copper oxygen carrier hemocyanin is discussed in light of these results.

Paramagnetically shifted resonances in 1H NMR spectra of ribonucleotide reductase from Escherichia coli.

The 400-MHz 1H NMR spectra of the subunit B2 of ribonucleotide reductase from Escherichia coli show paramagnetically shifted resonances at 24 ppm (exchangeable protons) and at 19 ppm (nonexchangeable protons). The protein contains an antiferromagnetically coupled dimeric iron center and a tyrosyl free radical. The paramagnetically shifted resonances must be due to the iron center, since they remain essentially unchanged in protein B2 with and without free radical. In analogy with recently published results for hemerythrin from Phascolopsis gouldii, which has a similar iron center, the 24-ppm resonance is suggested to arise from histidine ligands to the iron ions.

A cytochrome b5 from erythrocytes of Phascolopsis gouldii. One component of a potential system for reduction of methemerythrin in vivo

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA cytochrome b5 from erythrocytes of Phascolopsis gouldii. One component of a potential system for reduction of methemerythrin in vivoRonald E. Utecht and Donald M. Kurtz Jr.Cite this: Inorg. Chem. 1985, 24, 26, 4458–4459Publication Date (Print):December 1, 1985Publication History Published online1 May 2002Published inissue 1 December 1985https://doi.org/10.1021/ic00220a007RIGHTS & PERMISSIONSArticle Views25Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (264 KB) Get e-Alerts Get e-Alerts

Nitric oxide adduct of the binuclear iron center in deoxyhemerythrin from Phascolopsis gouldii. Analog of a putative intermediate in the oxygenation reaction

Nitric oxide adduct of the binuclear iron center in deoxyhemerythrin from Phascolopsis gouldii. Analog of a putative intermediate in the oxygenation reaction

The interaction of hemerythrin with sodium dodecyl sulfate and the release of iron from the product with desteferrioxamine B

The interactions with sodium dodecyl sulfate (SDS) of methemerythrin, the anionic derivatives and oxyhemerythrin from Phascolopsis gouldii have been examined at 25°C, I = 0.5 M and pH 6.3 and 7.8. Absorbance changes in the 350–500 nm range were used to monitor the rates. The denaturation is slow ( k = 10 −2–10 −3 s −1) and only slightly dependent on SDS concentration. Perchlorate is a very effective inhibitor of the SDS reaction with methemerythrin, and it is concluded that rapid binding of SDS near to the cysteine-50 site is an essential to unfolding. Myohemerythrin (from Themiste zostericola) and the monomeric N-ethylmaleimide derivatives of methemerythrin from P. gouldii and T. zostericola, in contrast, react rapidly with SDS. The products from denaturing of all proteins appear similar, having reduced α-helix content, very small absorbance in the 350–500 nm region and loss of anion or oxygen binding capacity. They do, however, retain the two irons, which can readily be removed with desferrioxamine B.

The kinetics of oxidation of hemerythrin species—linear free energy relationships

The second-order rate constants (M −1sec −1, 25°C, pH 8.2, I = 0.15 M) for the oxidation to (semi-met) 0of deoxyhemerythrin from Phascolopsis gouldii (P.g.) and Themiste zostericola (T.z.) have been determined for Fe(CN) 5(4-NH 2py) 2− (3.6 × 10 4 T.z.,2.8 × 10 2P.g.),Fe(CN) 5NH 3 2−(2.4 × 10 4 T.z.), Fe(CN) 6 3− (1.0 × 10 5 T.z.,1.4 × 10 2P.g.), Fe(CN) 5PPh 3 2− (7.3 × 10 5T.z.), and Fe(CN) 4dipy- (~6 × 10 6 T.z.,7.5 × 10 4 P.g.). Corresponding rate constants for the oxidation of (semi-met) R to met are: Fe(CN) 5(4-NH 2py) 2− (1.2 × 10 3 P.g.), Fe(CN) 6 3− (3.4 × 10 5 T.z., 4.5 × 10 Fe(CN) 5PPh 3 2− (4.4 × 10 4P.g.), Fe(CN) 4dipy − (1.7 × 10 5P.g.), and Coterpy 2 3+ (5.1 P.g.) The rates of oxidation of deoxy- and (semi-met) R myohemetythrin by Fe(CN) 6 3− were too rapid for stopped-flow measurement. The Marcus relationship for cross-reactions was successfully applied to these data.

Oxidation state of binuclear iron site in azidosemimethemerythrin

Electron paramagnetic resonance spectra of azidosemimethemerythrin (from Phascolopsis gouldii ) have been integrated to find the total number of spins per monomer unit. The value observed, 1.0 ± 0.1 spins per Fe 2 pair, confirms the assignment of a hybrid oxidation state, Fe IIFe III, to each site.

Reduction potentials of various hemerythrin oxidation states

The reduction potentials of the couple methemerythrin/(semi-met) r have been determined spectrally, using 2,6-dichlorophenolindophenol as a redox partner, for monomer and octamer from Themiste zostericola and for octamer protein from Phascolopsis gouldii. The reduction potentials of the couple (semi-met) o/deoxy have been determined spectrally, using Fe(III) cyanocomplexes, for monomer and octamer protein from Themiste zostericola. The values of other potentials, (met/(semi-met) o and (semi-met) r /deoxy), have thus been deduced as well as equilibrium constants for the (semi-met) r α (semi-met) o conformational changes for hemerythrins from Themiste zostericola.

X-ray absorption spectroscopy of the dimeric iron site in azidomethemerythrin from Phascolopsis gouldii.

X-ray absorption spectroscopy has been used to study the dimeric iron center in azidomethemerythrin from Phascolopsis gouldii. Absorption edge data confirm that the two iron atoms are present as Fe(III) and suggest a hexa-coordination site for each of the iron atoms. The extended x-ray absorption fine structure (EXAFS) analysis provides direct structural evidence of a mu-oxo bridge between the two iron atoms at an average Fe-O distance of 1.71-1.76 A. Analysis using a multiple-scattering formalism calculates upper limits of 165 degrees for the Fe-O-Fe bridging angle and 3.38 A for the Fe-Fe distance. This result agrees with current crystallographic models being determined by refinement of structures of two azidomethemerythrins.

Properties and distribution of a chymotryptic-like enzyme from the sipunculan, Phascolopsis gouldii

Homogenates of digestive tract, digestive fluid extracts, and a partially purified enzyme from Phascolopsis gouldii demonstrated activities toward N-benzoyl-L-tyrosine ethyl ester (BTEE) and N-glutaryl-L-phenylalanine p-nitroanilide (GPANA) at pH 7.8. The chymotryptic component showed optimal BTEE activity from pH 7.7 to 8.2 and was found to be stable at pH 5.0–9.0 with immediate and irreversible loss of activity at pH 4.0. BTEE activity was inhibited by L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), phenylmethylsulfonyl fluoride (PMSF), and phenothiazine-n-carbonyl chloride (PCC) and demonstrated affinity for 4-phenylbutylamine on a Sepharose 4B column. The molecular weight of the digestive fluid component as determined by gel chromatography was approximately 23 400. Gel chromatography of the eluant demonstrating pheynlbutylamine affinity yielded additional low molecular weight components. The posterior loop, which forms the junction between descending and ascending intestine, demonstrated highest BTEE and GPANA activities. In contrast, secretory cells are concentrated in the anterior descending intestine and granules appear to be emitted as an apocrine secretion.