Theodore T. Herskovits

Structural Stability and Solvent Denaturation of Myoglobin

As judged from the midpoints of the denaturation transition of 31 water-miscible alcohols, ureas, and amides, the effectiveness of these denaturing agents on sperm-whale myoglobin increases with increasing chain length and hydrocarbon content, as expected in view of the disorganization of the hydrophobic interior of this protein. Increase in the hydroxyl content, blocking of the functional amino groups of the ureas and amides by alkyl substitution, and branching of the hydrocarbon portion of the denaturants are of less importance in determining the effectiveness of the denaturants.

Recent aspects of the subunit organization and dissociation of hemocyanins

1. The hemocyanins of the arthropod phylum are built of multiples of hexamers consisting of 1,2,4,6 and 8 of such basic assemblies. Their molecular weights range from about 0.45 x 10(6) to 3.9 x 10(6) daltons. The basic hexameric unit consists of bean-shaped monomers organized in the form of two layers of trimers placed on top of one another. The subunits are heterogeneous, in most cases consisting of four or more electrophoretically different polypeptide chains. 2. Molluscan hemocyanins have an entirely different structure and pattern of assembly from the arthropodan hemocyanins. The basic assembly of the molluscan hemocyanins are decamers organized in the form of right-handed cylinders approximately 300 A in diameter and 140-190 A in height. Different species have one, two and sometimes more than two such assemblies forming correspondingly longer cylindrical particles with molecular weights ranging from about 3.3 x 10(6) to 13 x 10(6) daltons. Cephalopod and chiton hemocyanins consist of single decameric particles, while gastropods have hemocyanins organized of di-decamers or higher assemblies. The subunits of these hemocyanins are elongated protein chains with seven or eight folded globular domains, each housing a binuclear copper center capable of binding and delivering oxygen. 3. The dissociation behavior of the arthropod hemocyanin hexamers and di-hexamers with the hydrophobic urea series of reagents suggest polar and ionic interactions as the main sources of stabilization of the hexamers and the hexamer to hexamer contacts within the di-hexamers. 4. Dissociation studies with the same urea probes with the molluscan hemocyanins, however, suggest a different pattern of stabilization. The stabilization of the decamer to decamer contacts within the gastropod di-decamers appear to be predominantly polar and ionic with relatively few hydrophobic interaction sites. The dimer contacts within the decamers and the monomer to monomer contacts within the dimers observed in the octopus and chiton hemocyanins appear to be predominantly hydrophobic in nature. 5. The urea and the pH dissociation profiles of the single decameric assemblies of some of the octopus and chiton hemocyanins investigated by light-scattering molecular weight methods, have been fitted using either a two-species, decamer to dimer and decamer to monomer scheme of subunit dissociation or a three-species, decamer to dimer to monomer scheme of dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)

Solution studies on heme proteins: circular dichroism and optical rotation of Lumbricus terrestris and Glycera dibranchiata hemoglobin.

Abstract The circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of the multi-chain hemoglobin of the earthworm, Lumbricus terrestris, are presented and compared to the spectra of the single chain and four-chain Glycera dibranchiata and human A hemoglobins in both the ferro and ferri heme oxidation states. In Fe2+ oxy form L. terrestris hemoglobin exhibits positive dichroic bands at 258,412, and 560 nm and negative bands at 288,330, 520, and 578 nm. Except for the presence of a positive CD band at 577 nm, corresponding to the 578 nm band of L. terrestris oxyhemoglobin, human oxyhemoglobin has similar CD spectra in the same near-ultraviolet to visible wavelength region. On the other hand, all the resolved dichroic bands or extrema of G. dibranchiata hemoglobin at 280, 295, 345 and 540 nm are negative, save for the last band at 575 nm, which is positive. These differences seem to result from the changes in the heme environment and the geometry, and coordination state of the central heme iron in these proteins. While the changes in the oxidation state to the Fe3+ form of these hemoglobins seem to produce significant changes in the position and amplitude of most of the observed CD bands, including a change in the sign of L. terrestris cyanomethemoglobin at 525 and 575 nm, the largely negative character of the CD spectra of the G. dibranchiata cyanomethemoglobin (with negative CD bands shifted to 270, 297, 364, 427, and 548 nm) seems to be preserved. The ORD and CD spectra in the far-ultraviolet, peptide absorbing region are found to be largely unaltered on changes in oxidation states and ligand binding. The CD spectrum of L. terrestris oxyhemoglobin is thus characterized by CD bands centering at 218, 208 and 193 nm, with mean residue ellipticities [Θ]λ = −15 750, −14 900, and 33 100 deg. · cm2 · dmole−1, and the ORD spectrum with extrema at 233 and about 200 nm with mean residue rotations, [m′]λ of −5430 and 33 000 deg. · cm2 · dmole−1. The corresponding CD and ORD amplitudes of G. dibranchiata oxyhemoglobin are found to be, respectively, [Θ]λ = −24 350, −23 100, and 61 600, and [m′]λ = −9180, and 48 400 deg. · cm2 · dmole−1. Estimates of apparent helix content obtained with the recently published ORD and CD reference parameters by Chen et al., based on X-ray structural and solution data of various proteins, indicate rather low helical folding of 33 to 45% for L. terrestris hemoglobin and 70 to 75% helix for G. dibranchiata hemoglobin. The latter estimate is similar to that obtained on human hemoglobin A. The possibility that the low estimates of helix content of L. terrestris hemoglobin are caused by spectral distortions and absorption flattening due to Mie scattering of this high molecular hemoprotein was considered, and ruled out, largely on the basis of light scattering and concommitant ORD and CD measurements as a function of pH. Light scattering measurements at 630 nm confirmed the mol. wt of approx. 3.1 · 106 at pH 7.0. Dissociation of the relatively large subunit organization of this protein in the alkaline region, at pH 10.1 to 10.4, to about a tenth to a twelfth of the original molecular weight was found to have relatively little or no effect on ORD and CD spectra. No increases in the amplitudes and significant shifts in the wavelengths of the 233 and 200 nm ORD and 218, 208, and 193 nm CD band were found, suggesting that spectral distortions are of minor importance regarding the ORD and CD spectra, and estimates of helix content of L. terrestris hemoglobin.

Light scattering studies of the quaternary structure and subunit dissociation of proteins: The use of hydrophobic solutes and salts as probes☆

Abstract The use of hydrophobic reagents and salts on the molecular weight behavior of subunit proteins, such as the four-chain human and the multichain earthworm hemoglobins, is described in relation to the forces that stabilize their quaternary structure in solution, and the nature and number of amino acids located at the contact areas of their subunits. The equations derived for analysis of light scattering dissociation data are described together with the use of binding and Setschenow constants of the probe molecules or dissociating reagents. For human hemoglobin A the dissociation to half molecules, with several alkylureas, aliphatic acid salts, and some members of the Hofmeister salt series, gave estimates for the number of amino acids at the contact areas of the subunits within the ranges of 19 and 27 amino acid residues suggested by the X-ray crystallographic structure of horse and human hemoglobin.

HIGHER ORDER ASSEMBLIES OF MOLLUSCAN HEMOCYANINS

1. The hemocyanins of the Fissurellidae, Naticidae and Melongenidae families of marine gastropods as well as some other molluscs including some members of the Opistobranchia and Bivalvia groups have hemocyanins which exist in solution as tri-decameric and mixed, multi-decameric aggregates characterized by sedimentation coefficients close to 100 S, 130 S, 150 S, 170 S and 200 S to 230 S. 2. The particle masses of the molluscan hemocyanins appear to be integral multiples close to 4.4 x 10(6) daltons. Thus, particle mass values of 4.47 x 10(6), 8.67 x 10(6) and 13.40 x 10(6) daltons were obtained for representative decameric, di-decameric, and tri-decameric components of Stenoplax conspicua, Fasciolaria tulipa and Euspira (Lunatia) heros hemocyanins. For Busycon contrarium, a gastropod with a mixed multidecameric hemocyanin, scanning transmission electron microscopic (STEM) measurements gave particle masses ranging from 8.89 x 10(6) and 13.20 x 10(6) for the di- and tri-decameric components to 38.87 x 10(6) and 43.40 x 10(6) daltons for highest nano- and deca-decameric aggregates. 3. The electron microscopic images of both uranyl acetate-stained and unstained specimens of hemocyanin aggregates indicate a non-random mode of assembly of the multi-decameric particles. This is most apparent from the electron micrographs of the moon snail hemocyanins. The tri-decameric and tetra-decameric particles seem to be assembled from a single di-decameric unit of the Mellema and Klug arrangement, with the collar ends facing outward, to which decameric units have been added from one or both ends, in a unidirectional tail-to-head to tail-to-collar manner. Consequently, all the aggregates including the higher, Melongenidae polymers have the appearance of closed cylinders terminating with the collar ends. 4. The radial distribution of the end-on views of the hemocyanin of the moon-snail Calinatioina oldroydii, show that the radial mass drops to zero at the center of the cylindrical particles consisting of one, two, or three decamers. This suggests that no caps are present at the ends of the hemocyanin particles which would inhibit or terminate their linear assembly. 5. The light-scattering behavior of B. contrarium and Marisa cornarietis hemocyanins examined as a function of increasing reagent concentration using the hydrophobic urea and Hofmeister salt series of reagents, show distinct aggregation and increase in molecular weights at low concentrations of reagent. Together with the stabilizing influence of Mg2+ and Ca2+ ions, this suggests polar and ionic stabilization of the inter-decameric contacts between the central di-decamers and the added decameric units of the higher aggregates of molluscan hemocyanins.(ABSTRACT TRUNCATED AT 400 WORDS)

Subunit structure and dissociation of Callinectes sapidus hemocyanin

The hemocyanin of the blue crab, Callinectes sapidus has two major components sedimenting with approximate sedimentation coefficients of 17 S and 25 S. Molecular weight data based on light scattering and sedimentation equilibrium measurements at pH 7.8 suggest that the two components have molecular weights of approximately 450 000 and 900 000 in the presence of stabilizing Ca2+. In the absence of Ca2+, the molecular weights are found to be about 5% lower, suggesting some dissociation of the hemocyanin components. Circular dichroism and optical rotatory dispersion measurements in the far-ultraviolet region gave nearly identical spectra for the two components. Based on the reference parameters of Chen et al. (Chen, Y.H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131 and Chen, Y.H., Yang, J.T. and Chan, K.H. (1974) Biochemistry 13, 3340--3359), estimates of 16--20% alpha-helix, 40--60% beta-structure, and 30--40% random organization were obtained for the two hemocyanin components. Exposure to 6 M Gdn HCl gave light scattering molecular weights of approx. 68 000 and 77 000, which is close to one-sixth of the molecular weight of the 17 S component. These results support the view that the two components of C. sapidus hemocyanin share the hexameric and dodecameric organization common to arthropod hemocyanins. The salts of the Hofmeister series and the ureas are found to dissociate the dodecameric component with the former exhibiting the usual order of effectiveness of NaCl, NaBr, NaI, and NaClO4 dissociation, while the ureas show an inverse order of decreasing effectiveness in going from urea to methyl-, ethyl- and propylurea. This suggests that polar and ionic interactions are relatively more important than hydrophobic interactions for the stabilization of the dodecameric form of C. sapidus hemocyanin. The dissociation behavior of the 17 S hexameric species by GdnHCl in the 0--1.5 M concentration region (where essentially no denaturation occurs), based on light scattering molecular-weight measurements, is satisfactorily accounted for by equations describing the dissociation of hexamers to monomers.

Solvent denaturation of globular proteins Unfolding by the monoalkyl- and dialkyl-substituted formamides and ureas

The effects of the monoalkyl and dialkyl-substituted formamide series of denaturants on the native conformation of sperm whale myoglobin, horse heart cytochrome c, and Glycera dibranciata (single chain) hemoglobin have been investigated by spectral measurements in the Soret region (409 and 422 nm) and optical rotation measurements (265nm). The effectiveness of these two classes of protein denaturants is similar to the other straight-chain compounds of the urea, amide, and alcohol classes, examined in previous investigations from our laboratory. Their denaturing effectiveness is found to increase with increasing chain length or hydrocarbon content of the substituent alkyl groups. Application of the Peller and Flory equation to the denaturation data of the formamides shows that both the polar and the nonpolar group contributions to the protein-denaturant interactions have to be taken into account in order to correctly predict the observed denaturation midpoints. Additivity of the hydrophobic, KHø, and the polar, Kp, group contributions to the binding constants, KB = nKHø + Kp, with n = 1 or 2 for the mono- of the di-alkyl substituted denaturants gave best account of the experimental data. The KHø values used were based on free energy transfer data of various alkyl groups or the Scheraga-Nemethy theory of hydrophobic bonding. The assumption of group contributions of the denaturant to KB were also applied to the denaturation data of the unsubstituted amides and some examples of the monoalkyl and symmetrically substituted dialkyl ureas, taken from the literature.

Solvent perturbation studies of heme proteins and other colored proteins. I

Abstract The solvent perturbation technique of difference spectroscopy has been applied and extended to the study of the location of the aromatic tryptophyl and tyrosyl residues in the heme proteins. Based on experiments on unfolded hemoglobin and myoglobin and their apoproteins in 8 m urea and acid solutions, and their model compound analogs, corrections for the heme-contribution to the difference spectra in the 270–295-mμ aromatic region have been suggested, and their validity and limitations have been described and tested. These corrections are based on extrapolations dictated by the difference spectra of the nonaromatic, neighboring bands, or the γ-and δ-bands in the case of the heme proteins. The location of the tryptophyl residues in horse heart cytochrome c, beef liver catalase, sperm whale myoglobin, and horse hemoglobin, in the ferri state have been studied, using 20% ethylene glycol, glycerol, sucrose, and 90% deuterium oxide as perturbants. The data obtained on cytochrome c suggest that the single tryptophyl residue in this protein is about 55 to 68% exposed. In the case of beef liver catalase, a much larger subunit protein, a substantially greater fraction of the approximately 40 tryptophyls appear to be buried. In this protein 35 to 40% of the tryptophyl seem to be affected by the perturbing influence of solvent. Comparison of the hemoglobin and myoglobin data indicate a somewhat greater degree of burial of the tryptophyls in hemoglobin than in myoglobin (in myoglobin there are two tryptophyls whereas in hemoglobin there are six such groups). A more detailed discussion and analysis of the horse hemoglobin data based on studies with six perturbants, as well as the perturbation data for five other mammalian hemoglobins obtained with 20% ethylene glucol, is presented in the accompanying paper.

The effects of salts and ureas on the subunit dissociation of concanavalin A

The effects of various neutral salts of the Hofmeister series and the urea series on the subunit structure and dissociation of concanavalin A were investigated, employing light-scattering molecular weight methods. Concanavalin A and its isolated intact chain tetramers prepared by the method of Cunningham and co-workers (Biochemistry 11 (1972), 3233–3239) are found to dissociate rapidly and reversibly to dimers. The higher members of the Hofmeister series and GdnCl are found to be the most effective dissociating reagents, with the former salts exhibiting the usual trend of increasing effectiveness: F− < CI− < Br− < ClO4−, SCN−, I−. The alkyl ureas are found to be generally less effective dissociating agents than GdnCl and the higher members of the Hofmeister salt series. This suggests that, relative to polar and ionic interactions, hydrophobic effects have a less dominant stabilizing influence on the dimer-dimer interactions, generating the tetramers in solution. The combined effects of pH and urea on the tetramer to dimer dissociation of concanavalin A in the pH 6.0 to 7.5 region was also investigated. The light-scattering molecular weight data could be fitted with either one or two protonating groups per monomer, depending on the protein preparation. The pK values of 6.7, or 6.4 and 6.7, used to fit the dissociation data suggest that probably histidine residues 51 and 121 (assigned by Senear and Teller (Biochemistry 20 (1981), 3076–3083)) are involved in the proton-linked association-dissociation reaction observed at pH 6.0 to 7.5. The possibility that one of the two protonating groups is glutamic acid residue 192 has also been considered.

Subunit dissociation of Busycon canaliculatum hemocyanin

The hemocyanin of the channeled whelk, Busycon canaliculatum, is a multisubunit protein with a molecular weight close to 9 X 10(6). The increase in pH above neutrality and the addition of 0-5 M urea and 0-2 M GdnHCl is found to dissociate the whole molecules to half-molecules and smaller dimeric and monomeric fragments of one-tenth and one-twentieth mass of the parent hemocyanin. The molecular weight transitions investigated at constant protein concentration of 5 X 10(-2) g X l-1 show no clearly discernible plateau regions, where essentially only half-molecules and one-tenth molecules are present. The ultracentrifugation patterns in much of the dissociation region produced by urea at pH 6.9 suggests the presence of three distinct components consisting of whole molecules, half-molecules and largely one-tenth molecular weight fragments. At pH 8.2 and higher, where whole molecules are largely absent, the effects of urea on the dissociation of half-molecules to tenths and tenth-molecules to twentieth molecule was investigated by means of light scattering. Analysis of the urea data based on a decamer to dimer and dimer to monomer scheme of dissociation used in our earlier studies gave apparent estimates of about 90 amino acid groups at the contact areas of the dimers in the half-molecules and 110 groups at the monomer contacts forming the dimers. The latter relatively large estimate of groups suggests that the dissociation of the tenth molecules or dimers must occur by longitudinal splitting of the contact areas along both the folded domains and the connecting chain segments of the twentieth molecules. Circular dichroism, absorbance and viscosity data suggest that the secondary structure and conformation of the folded domains of the hemocyanin subunits are largely retained at both high pH and in 3-8 M urea solutions. The molecular weights at pH 9.0-10.6 and in 3-8 M urea are found to be (4.2-4.7) X 10(5), close to one-twentieth of the mass of the parent hemocyanin. Denaturation and unfolding of the subunit domains is observed between 3 and 6 M GdnHCl solutions, as evidenced by the abolition of the characteristic copper absorbance in the neighborhood of 346 nm and the relatively pronounced changes in circular dichroism at 222 nm and intrinsic viscosity. The further decrease in molecular weights to about (2.6-3.2) X 10(5), below one-twentieth of the mass of hemocyanin suggests the presence of hidden breaks or scissions in the polypeptide chains suffered during isolation, which become exposed as a result of complete unfolding in GdnHCl solutions.(ABSTRACT TRUNCATED AT 400 WORDS)