Near-ultraviolet Circular Dichroism Research Articles

Stepwise dissociation/denaturation and reassociation/renaturation of bovine α-crystallin in urea and guanidine hydrochloride: Sedimentation, fluorescence, near-ultraviolet and far-ultraviolet circular dichroism studies

Quaternary, tertiary and secondary structure changes of calf α-crystallin were studied as a function of urea and guanidine hydrochloride concentration at pH 7·3 and 20°C. Sedimentation velocity analysis shows that native α-crystallin (19 s, 850 000 M r) dissociates in a step-wise fashion to single subunits (1·4 s, 20 000 M r). Complete dissociation is reached above 5 m-urea or 2·5 m-guanidine hydrochloride. The most stable dissociation intermediate (12 s) is observed around 2–3 m-urea or 1–1·5 m-guanidine hydrochloride. Fluorescence and near-ultraviolet circular dichroism analyses indicate that the exposure of aromatic residues is correlated with step-wise dissociation. Far-ultraviolet circular dichroism analysis shows that unfolding of subunits occurs over a very wide range of denaturant concentrations. Unfolding begins as subunits are released from the 12 s dissociation intermediate and may not be complete even in 8 m-urea or 6 m-guanidine hydrochloride. Dissociation of native α-crystallin to 12 s intermediate is irreversible, but subsequent dissociation steps are reversible. Dissociated and denatured subunits can be reassembled in a 12 s ‘reassociated α-crystallin’ molecule which is identical to the 12 s dissociation intermediate in nearly every aspect of secondary, tertiary and quaternary structure. The results are discussed in relation to our earlier proposed three-layer subunit model of calf α-crystallin and are found to support this model.

Reconstitution of lactic dehydrogenase after acid dissociation. The yield of reactivation is determined by conformational rearrangements of the dissociated monomers.

Reconstitution of lactic dehydrogenase from pig muscle after dissociation at acidic pH produces native tetramers and inactive aggregates, both of which have been previously described with respect to their structural properties and the kinetics of their formation. The ratio of the two fractions is determined by the relative rates of reassociation and aggregation [G. ZettlmeißI, R. Rudolph and R. Jaenicke (1979) Biochemistry 18, 5567–5571]. The present study is concerned with the influence of residual structural elements within the acid‐denatured monomers on the process of reconstitution.Long incubation at low pH leads to a marked decrease of the rate and yield of reactivation, caused by conformational changes within the partially unfolded monomers. These slow rearrangements are reflected by slight changes of the far‐ultraviolet circular dichroism of the acid‐dissociated monomers, indicating a decrease in helix content from 30 observed immediately after the transfer to low pH, to 25% after completion of the acid transition. The unchanged near‐ultraviolet circular dichroism and fluorescence properties prove local structural alterations to be of lesser importance compared to the changes of the residual backbone folding. Varying the experimental conditions during denaturation proves the slow decrease in the rate and yield of reactivation, as well as the changes in the far‐ultraviolet circular dichroism to be enhanced with incubation time and temperature (0–30°C). The first‐order rate constants of the decrease in the reactivation yield after incubation in 1 M glycine/H3PO4 pH 2.3, 0.1 M sodium phosphate pH 2.3, and 1 M H3PO4 were found to be in the same time range as the respective changes in the far‐ultraviolet circular dichroism.Apart from the similarity of the rate constants, the activation energy for both the acid induced changes of the yield of reactivation and the far‐ultraviolet circular dichroism is found to be identical (80 kJ/mol).The effects of low pH on the reconstitution of lactic dehydrogenase can be eliminated by complete disruption of the structure of the polypeptide chain, e.g. by adding 6 M guanidine · HCl. This proves unambiguously that the above‐mentioned structural rearrangements at acid pH are responsible for the observed decrease of the rate and yield of reactivation.

ChemInform Abstract: CHIROPTICAL PROPERTIES OF PROTEINS. I. NEAR‐ULTRAVIOLET CIRCULAR DICHROISM OF F RIBONUCLEASE S

Chemischer InformationsdienstVolume 12, Issue 8 Physical Organic Chemistry ChemInform Abstract: CHIROPTICAL PROPERTIES OF PROTEINS. I. NEAR-ULTRAVIOLET CIRCULAR DICHROISM OF F RIBONUCLEASE S W. J. GOUX, W. J. GOUXSearch for more papers by this authorT. M. JUN. HOOKER, T. M. JUN. HOOKERSearch for more papers by this author W. J. GOUX, W. J. GOUXSearch for more papers by this authorT. M. JUN. HOOKER, T. M. JUN. HOOKERSearch for more papers by this author First published: February 24, 1981 https://doi.org/10.1002/chin.198108061AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume12, Issue8February 24, 1981 RelatedInformation

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.

The role of lysine-41 of ribonuclease A in the interaction with RNase inhibitor from human placenta.

3-N-Carboxymethyl-His-12 and 1-N-carboxymethyl-His-119-RNase A bind to the naturally occurring RNase inhibitor, isolated from human placenta, 1.3 and 3.6 times, respectively, more strongly than does native RNase A. Near-ultraviolet circular dichroism measurements indicate that the conformational change which occurs upon carboxymethylation of either of the active site histidine residues appears different from that which the protein undergoes upon binding of substrate of a substrate analogue. Specific carboxymethylation of Lys-41 of RNase A decreased the strength of the interaction between the enzyme and the RNase inhibitor to about 12% of the initial value. The near-UV CD spectra of Cm-Lys-41-RNase A and of acetimidyl-RNase A (9.3 lysines modified) and carbamylated RNase A (3.0 lysines modified), which also have weaker interactions with RNase inhibitor of 25% and 10%, respectively, show a negative [theta]MRW identical to that of native RNase A at 275 nm but are altered in the positive [theta]MRW at 240 nm. The CD measurements suggest that one or more tyrosine residues of RNase A may be involved in the interaction with inhibitor. The effects of pH and salt concentration suggest that a major part of the protein-protein interaction is probably through nonpolar forces. The strengths of interactions between the inhibitor and pancreatic RNases from several species were very similar. Since Tyr-92 is the only tyrosine residue retained in all of the species studied, this residue may have a key role in the nonpolar interaction. The data presented herein suggest that the interaction between RNase A and the inhibitor involves the positively charged epsilon-NH2 group of Lys-41 of RNase A. This interaction could result in the inactivation of the enzyme.

Chiroptical properties of proteins. I. Near-ultraviolet circular dichroism of ribonuclease S

Chiroptical properties of proteins. I. Near-ultraviolet circular dichroism of ribonuclease S

THERMOLYSIN AND BACILLUS SUBTILIS NEUTRAL PROTEASE

A comparative study on thermolysin from Bacillus thermoproteolyticus and neutral protease from Bacillus subtilis involving (far- and near-ultraviolet circular dichroism (CD) and immunological techniques is reported. These enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors. The far-ultraviolet CD spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of ellipticity, however, have been observed. Estimates of secondary structure obtained by quantitation of the far-ultraviolet CD spectra indicated a higher helicity of neutral protease relative to thermolysin. In the presence of ethylenediaminetetraacetic acid, which removes calcium and the functional zinc ion from the metalloenzymes, neutral protease is immediately denatured, whereas thermolysin maintains a globular structure, although thermolabile. On the other hand, the zinc-specific chelating agent tetraethylenepentamine does not have measurable effects on the conformation and conformational stability of either protein. Marked higher stability to temperature and guanidine hydrochloride were observed for thermolysin as compared with neutral protease, as indicated by monitoring conformational transitions with CD measurements at 220 nm. Antisera prepared in rabbits using thermolysin as immunogen do not cross-react with neutral protease, indicating differences of surface structure between the two proteins. On the basis and limitations of the techniques employed, it is proposed that the two sequentially and functionally homologous metalloendopeptidases may have similar conformations at specific regions (active and binding sites, at least) of the polypeptide chain essential for biological function, while some variability in the structure of other regions may be tolerated.

Evolutionary and developmental differences in delta-crystallin from bird and reptile lenses Circular dichroism and fluorescence studies

1. 1. Delta-crystallin has been isolated by preparative isoelectric focusing from embryonic and/or adult lenses of the chicken, quail, turkey, duck, caiman and gecko. The secondary and tertiary structures of the delta-crystallins were examined by circular dichroism (CD) spectroscopy in the near- and far-ultraviolet and by fluorescence spectroscopy. 2. 2. Age-dependent differences were observed in the structure of delta-crystallin in the chicken and the turkey. Adult delta-crystallin had less alpha-helical structure when examined by far-ultraviolet CD spectroscopy, less rotatory strength in the near ultraviolet and less fluorescence when excited at 280 nm than embryonic delta crystalline. Despite these spectral differences, embryonic and adult chicken delta-crystallins behaved similarly when subjected to sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, gel filtration and isoelectric focusing in a sucrose density gradient. 3. 3. Far-ultraviolet CD spectroscopy indicated that delta-crystallin from the embryonic chicken, quail and turkey was approximately 80% alpha-helical, while that from embryonic duck was about 40% alpha-helical. By contrast, delta-crystallin from the adult chicken and turkey had about 30% alpha-helix, and that from adult caiman and gecko had about 30% and 10% alpha-helic, respectively. The near-ultraviolet CD spectra of the embryonic chicken, quail and turkey were similar, while that of the embryonic duck was related in structure but had less rotatory strength and resembled the profile of delta-crystallin from adult chicken, turkey and gecko. The fluorescence spectra of delta-crystallins from all species examined had emission maxima at 315 nm after excitation at 280 nm and 295 nm. There were species-dependent and age-dependent differences in intensity of the fluorescence spectra. Taken together, the data indicate considerable conservation of the conformation of delta-crystalline throughout evolution.

Circular dichroism study of the solution conformation of luteinizing hormone releasing hormone

A systematic investigation has been made into the circular dichroic behavior of luteinizing hormone releasing hormone and its peptide fragments and deletion analogues. The results are interpreted to mean that the hormone exists in solution as an ensemble of conformers with different sensitivities to temperature and solvent composition. The far-ultraviolet circular dichroic spectra exhibited by the hormone under different experimental conditions can be simulated satisfactorily by the weighted addition of the spectra of its aliphatic- and aromatic-containing halves. However, the structure of the hormone is not simply the sum of its halves, since some conformational feature of the intact molecule perturbs the near-ultraviolet circular dichroism of its aromatic residues.

Subunits of RNA polymerase in function and structure. 7. Structure of premature core enzyme

The structure of premature core enzyme, an obligatory intermediate in both in vivo and in vitro assembly of Escherichia coli DNA-dependent RNA polymerase, was compared with that of native core enzyme. Though this assembled but inactive form of core enzyme harbors the gross conformation similar to that of native enzyme, minor and presumably local differences exist, which were identified by near-ultraviolet circular dichroism spectra, tritium-hydrogen exchange rate, protease sensitivity, intersubunit cross-linking rate by bifunctional reagents, sedimentation behavior, and elution profile from phosphocellulose. Taken together these results indicate that the core enzyme subunits are loosely associated in the premature core. The temperature-dependent maturation is required for the core subunits to be tightly associated, leading to the formation of structurally stable and functionally active RNA polymerase.

16] The interpretation of near-ultraviolet circular dichroism

Circular dichroism (CD) is an absorptive phenomenon. It is defined as the difference between the absorbances of left and right circularly polarized light. This chapter describes a scheme for calculating the CD spectrum of proteins of known three-dimensional structure. Rotating about covalent bonds to modify the conformation with recalculation of the spectrum for the new three-dimensional structure gives the dependence of the optical activity upon conformation. The protein Hamiltonian constructed in the chapter has as its basis set the wave functions of the separate, individual chromophores that are present in the macromolecule. In the wave functions, isolated chromophoric states are mixed with one another, expressing the fact that the electrons of each chromophore may perturb those of all others. To formulate the Hamiltonian that yields these interactions, the chapter establishes a naming and ordering convention for the states of the macromolecule. It also examines interpretive work on the side chains of tyrosine, tryptophan, and the disulfide. These are the major contributors to the near-UV CD of proteins. Phenylalanine and histidine are omitted partly because their contributions are generally much weaker and partly because the state of our understanding of them is less advanced. For each chromophore model, compounds are examined followed by protein studies where available.

Spectral changes in bovine factor X associated with activation by the venom coagulant protein of Vipera russelli.

Bovine Factor X is a zymogen involved in blood coagulation that is converted to activated Factor X in the presence of Ca(II) by the coagulant protein of Russell's viper venom. To monitor structural transitions in Factor X during conversion to activated Factor X, the ultraviolet absorption, fluorescence emission, and circular dichroism spectra of activated Factor X and Factor X were compared. The ultraviolet absorption difference spectrum in the aromatic region comparing activated Factor X and Factor X indicates minima at 292.5, 285, and 278 nm and a small maximum at 305 nm; these differences are due to tryptophan and tyrosine perturbations. The activation of Factor X at 25 degrees in the presence of 8.3 mM CaCl2 with the use of Factor X:venom coagulant protein in molar rations of 1500:1 yielded a time-dependent increase in this spectrum which was linear for about 60 min and which temporally paralleled the development of activated Factor X activity. The binding of Ca(II) to factor X or activated Factor X is associated with a red-shifted tryptophan difference spectrum; however, this perturbation appears to make only a small contribution to the total perturbation observed during Factor X activation. Solvent perturbation studies in 20% glycerol suggest that an average of 3.1 tryptophan residues and 9.0 tyrosine residues are exposed to solvent in Factor X in 8.3 mM CaCl2 at pH 7.4; an additional 0.5 tryptophan residue and tyrosine reside become exposed to solvent during activation of Factor X in 8.3 mM CaCl2. The activation of Factor X by the venom coagulant protein is associated with a small red shift in the intrinsic tryptophan fluorescence emission spectrum. Far- and near-ultraviolet circular dichroism spectroscopy detected no difference between Factor X and activated Factor X. In summary, the activation of Factor X to activated Factor X appears associated with exposure of tryptophan and tyrosine side chains previously buried within the protein and with minimal changes in the secondary structur. These results suggest that conversion of Factor X to activated Factor X involves functionally important, but structurally subtle, changes in the three-dimentional structure.

Bovine adrenal medulla troponin-C. Demonstration of a calcium-dependent conformational change.

A high affinity calcium-binding protein has recently been purified from the adrenal medulla (Kuo, I.C.Y., and Coffee, C.J. (1976) J. Biol. Chem. 251, 1603-1609). This protein is closely related in its chemical and physical properties to troponin-C (TN-C) of muscle tissue. Further examination of the adrenal medulla protein indicates that the removal of calcium is accompanied by a marked change in the conformation. This change in structure is similar, if not identical, to the calcium-dependent conformational change which has been described for skeletal muscle TN-C (Murray, A.C., and Kay, C.M. (1972) Biochemistry 11, 2622). The far ultraviolet circular dichroism spectrum of native adrenal medulla calcium-binding protein (AM-CBP) shows characteristic helical ellipticity bands at 222 and 207 nm. The helical content, as estimated from these data, is between 40 and 45%. Removal of calcium is accompanied by a change in ellipticity which corresponds to a decrease from 40 to 20% in the helical content. The near-ultraviolet circular dichroism spectrum shows negative dichroic bands at 262 and 268 nm which are characteristic of phenylalanine. These bands are relatively insensitive to changes in the calcium ion concentration. Sedimentation velocity studies likewise are indicative of a calcium-dependent structural alteration. The sedimentation coefficient of the native protein was observed to be 1.89 S. Similar measurements performed in the presence of 3 mM ethylene glycol bis(beta-aminoethyl ether) N,N, N', N'-tetraacetic acid (EGTA) gave a sedimentation coefficient of 1.50 S. The molecular weight, as determined by sedimentation equilibrium studies, was 16,000 regardless of whether the measurements were made in the presence of CaCl2 or EGTA. From the elution properties of AM-CBP on Sephadex G-100, the Stokes radius was observed to be 19.8 A in the presence of calcium and 21.9 A in the presence of EGTA. All of these changes which were induced by the addition of EGTA were completely reversible by the readdition of excess CaCl2. These data suggest that the removal of calcium from AM-CBP is accompanied by a pronounced conformational change which occurs without a molecular weight change. The decreased sedimentation coefficient, the increased Stokes radius, and the reduced helical content, which are observed for the apoprotein, indicate that removal of calcium results in a transformation from a compact symmetrical structure to one that is less ordered and more asymmetrical.

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.

Circular dichroism and conformational transition of Dolichos biflorus and Robinia pseudoacacia lectins

The conformation of the lectins from Dolichos biflorus and Robinia pseudoacacia was studied by means of circular dichroism (CD). It was found that N- acetyl- d-galactosamine induced significant changes in the near-ultraviolet CD spectrum of Dolichos lectin but was ineffective with the lectin from Robinia. Tyrosine and tryptophan chromophores were chiefly involved in this saccharide-lectin interaction. The far-ultraviolet CD spectra indicated that both lectins have a significant content of the pleated sheet conformation, but not much, if any, α-helix. The predominant conformation in these lectins is the aperiodic bend structure which is stabilised chiefly by hydrophobic interactions. This was ascertained by the effect of sodium dodecylsulfate on these proteins.

Conformational Studies of Ovine Lutropin (Luteinizing Hormone) and Its Native and Chemically Modified Subunits by Circular Dichroism and Ultraviolet Absorption Spectroscopy

Conformational features of ovine lutropin and its subunits were investigated as well as cyanogen-bromide-treated subunits and reconstitted dimers. Spectrophotometric titrations of tyrosine residues indicated that all the tyrosines could be ionized in lutropin and the subunits, but a least three of the tyrosines ionized abnomally in native hormone, two in the isolated α and none in the isolated β subunits. When unfolded at pH 2, the hormone exhibited a reversible difference spectrum corresponding to the exposure of approximately three buried tyrosine residues as estimated by thermal perturbation. Amplitudes of the difference spectra were correlated to the specific biological activity of the starting materials. The total number of tyrosine residues per mole of protein in agreement with sequence data was determined by magnetic circular dichroism. These spectra were shifted toward the shortest wavelengths, as for the ultraviolet absorption spectra, when the pH was lowered from 7 to 2. Near-ultraviolet circular dichroism was correlated to those results and both tyrosine residues and disulfide bridges were found to contribute to the negative 280-nm circular dichroic band. The foldings of lutropin and its isolated subunits were estimated in terms of only three structures, α-helix, β-sheet and aperiodic (irregular) structure from least-squares fitting of computed far-ultraviolet circular dichroism to experimental circular dichroism. The best fit for native hormone was 8%α-helix, 35%β-structure and 57% aperiodic. This fitting was in good agreement with the predicted conformation from amino acid sequence whith yielded 6%α-helix, 41%β structure and 52% aperiodic. During the acid unfolding, Which led to a biologically inactive hormone, most of the α-helix structure was lost and the β structure content was significantly decreased with a corresponding increase of aperiodic structure, indicative of a limited unfolding. Both subunits were found to participate to the unfolding. As compared with the predicted folding of the subunits in the native hormone, the isolated α subunit had essentially a lower β sheet content and the isolated β subunit a lower helical content. Refolded lutropin, obtained by raising the pH from 2.8 to 5.3, or recombined dimers from intact or cyanogen-bromide-treated subunits, which were biologically active, showed essentially the same conformational features as the native hormone. From the conformational criteria, cyanogen-bromide-treated subunits were indistinguishable from the intact subunits.

Circular dichroism changes in galactosyltransferase upon substrate binding;.

Circular dichroism studies with the galactosyltransferase isolated from bovine skim milk are described. Addition of UDP-galactose to the galactosyltransferase-Mn-2+ complex causes a decrease in the negative mean residue ellipticity in the 205-220-nm range and positive increases in the 265- and 275-290-nm ellipticity. These date are consistent with the view that a conformation change involving aromatic amino acid residues occurs upon the binding of UDP-galactose to the galactosyltransferase-Mn-2+ complex. No effects in the near-ultraviolet circular dichroism spectrum were observed upon the addition of UDP or glucose to the galactosyltransferase-Mn-2+ complex.

Influence of Prosthetic Groups on Protein Folding and Subunit Assembly: RECOMBINATION OF SEPARATED HUMAN α- AND β-GLOBIN CHAINS WITH HEME AND ALLOPLEX INTERACTIONS OF GLOBIN CHAINS WITH HEME-CONTAINING SUBUNITS

Abstract Reconstitution of the α and β subunits of human hemoglobin from separated α- and β-globin chains is described. The absorption and circular dichroism spectra, sedimentation coefficients, and sulfhydryl reactivities of the reconstituted subunits are compared with those of native subunits, which are regenerated from their p-mercuribenzoate chains by a novel procedure. The reconstituted subunits specifically recombine with the complementary native ones. Each property of the α subunit agrees, within experimental uncertainty, with the corresponding one of the subunit from which heme was never removed. Thus, markedly disordered and aggregated α-globin is restored to the highly ordered, monomeric α subunit upon recombination with 1 eq of hemin or hemin dicyanide. The reconstituted β subunit also compares well with the native subunit, except for a minor difference in a portion of the near-ultraviolet circular dichroism spectrum and a diminished sulfhydryl reactivity. Possible reasons for these differences are discussed. In the met form neither reconstituted subunit forms hemochromogen for several days, whereas the original met subunits become denatured within hours, probably owing to the necessity of exposing the latter, but not the former, to ferricyanide ion in producing the met forms. It is shown that while α- and β-globins do not combine at 4° (Yip, Y. K., Waks, M., and Beychok, S. (1972) J. Biol. Chem. 247, 7237), recombination does occur if either chain contains heme. The recombination produces half-filled (HF) molecules which are of two kinds, depending on which chain contains heme. The moleclar weights of these molecules are different. HFα, the intermediate prepared by mixing α subunits with β-globin, is a dimer; HFβ, the intermediate containing heme on its β chains, is a tetramer. No interchange of heme among chains occurs in the cyanmet forms in either molecule. In each, moreover, the conformation of the heme-free chain differs from that of the same chain when unbound. Both HF molecules bind 1 eq of heme, producing tetrameric hemoglobin. The most dramatic finding in this work is that the disordered α-globin can be refolded not only by binding heme but also by combining with a β subunit, although the refolding in the latter case is not as complete as in the former. These results show that the primary sequences of the globin chains generate unstable conformations, which are stabilized by combination with heme. They demonstrate also that refolding of a polypeptide chain can be induced by interaction with a neighboring subunit in an assembly process.

Conformational change of pepsin induced by its substrate N-acetyl- l-phenylalanyl- l-3,5-diiodotyrosine

Specific contributions of tyrosyl and of tryptophanyl residues can be distinguished in the near-ultraviolet circular dichroism spectrum of porcine pepsin. Upon addition of the dipeptide substrate, N-acetylphenylalanyl- l-3,5-diiodotyrosine, at pH values below 4.0, a change in the circular dichroic spectrum results, suggesting that in the presence of substrate the asymmetric environment of certain aromatic amino acid residues of the enzyme is altered. The changes observed are discussed in relation to the enzymatic function of pepsin.