Far-ultraviolet Circular Dichroism Spectra Research Articles

Regeneration of native ovalbumin conformation disrupted by citraconylation

Ovalbumin was reacted with a 960-fold molar excess of citraconic anhydride, and 91% of the ε-amino groups, representing 18 of the 20 lysine residues, were citraconylated. As detected by fluorescence and far-ultraviolet circular dichroic (CD) measurements, the modified protein displayed significant disruption of the native conformation. Treatment at pH 2.2 for 5 h resulted in the hydrolysis of 10 of the 18 citraconyl groups, but when subjected to the acid conditions for 12 h, all 18 modifying groups were removed. Electrophoretically, the 5-h and the 12-h acid-treated proteins were homogeneous and showed decreasing anodic mobility at pH 8.3; indeed, the anodic mobility of the 12-h acid-treated protein was identical to that of the native protein. Similarly, the 12-h acid-treated protein possessed conformational properties almost indistinguishable from the native protein. These properties included similar emission fluorescence spectra and far-ultraviolet CD spectra, similar resistance to undergoing helix-to-coil transition at pH 12.2. and identical urea unfolding curves, and thus identical urea transition mid-point of about 8.0 M. These observations indicate that the protein with all the ϵ-amino groups regenerated by acid treatment has the same conformational stability as the native ovalbumin.

A spectroscopic and conformational study of pertussis toxin

The conformation of native pertussis toxin has been investigated by secondary structure prediction and by circular dichroism, fluorescence and second-derivative ultraviolet absorption spectroscopy. The far-ultraviolet circular dichroic spectrum is characteristic of a protein of high beta-sheet and low alpha-helix content. This is also shown by an analysis of the circular dichroic spectrum with the Contin programme which indicates that the toxin possesses 53% beta-sheet, 10% alpha-helix and 37% beta-turn/loop secondary structure. Second-derivative ultraviolet absorption spectroscopy suggests that 34 tyrosine residues are solvent-exposed and quenching of tryptophan fluorescence emission has shown that 4 tryptophan residues are accessible to iodide ions. One of these tryptophans appears to be in close proximity to a positively charged side-chain, since only 3 tryptophans are accessible to caesium ion fluorescence quenching. When excited at 280 nm, the emission spectrum contains a significant contribution from tyrosine fluorescence, which may be a consequence of the high proportion (55%) of surface-exposed tyrosines. No changes in the circular dichroic spectra of the toxin were found in the presence of the substrate NAD. However, NAD did quench both tyrosine and tryptophan fluorescence emission but did not change the shape of the emission spectrum, or the accessibility of the tryptophans to either the ionic fluorescence quenchers or the neutral quencher acrylamide.

Structural properties of the microsomal triglyceride-transfer protein complex

The microsomal triglyceride-transfer protein (MTP), which catalyzes the transport of triglyceride and cholesteryl ester between membranes, is a complex composed of two proteins having apparent molecular weights of 58,000 and 88,000. The 58,000 molecular weight component of MTP has been identified as the multifunctional protein, protein disulfide isomerase (PDI). The multisubunit nature of MTP as well as the presence of PDI as one of the subunits distinguishes this protein from previously characterized lipid-transfer proteins. In this study, we have more clearly defined structural elements of MTP that may play important functional roles. The molecular weight of the transfer protein complex was determined to be 150,000 by sedimentation equilibrium experiments performed at three different speeds, suggesting that MTP is a complex of one PDI and one 88,000 molecular weight polypeptide (88K). Following SDS-polyacrylamide gel electrophoresis, the Coomassie Blue staining intensity of PDI in a known amount of MTP was compared to that of known amounts of a PDI standard. A 1 to 0.98-1.30 ratio of PDI to 88K was determined, confirming the 1:1 stoichiometry of MTP. The sedimentation coefficient (5.85) determined by analytical ultracentrifugation and the Stokes radius (47 A) determined by polyacrylamide gradient gel electrophoresis indicate that the 150,000 molecular weight MTP complex is asymmetric and/or has an unusually high water of hydration. PDI and 88K form a stable protein complex; there was no evidence of a dissociation-reassociation reaction occurring between the two components. Analysis of far-ultraviolet circular dichroism spectra revealed MTP has about 28% alpha-helical and 28% beta-structural content.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of amino acid ion pairs on peptide helicity

The three ER ion pairs in the peptide acetyl-W(EAAAR)3A-amide were replaced in turn with the ion pairs EK, EO, DR, DK, and DO, where O represents an ornithine residue. The far-ultraviolet circular dichroic spectra of the six peptides measured in 10 mM NaCl at pH 2 and 0 degrees C form a nested set having an isodichroic point at 203 nm of -17,000 deg cm2 dmol-1. The ellipticity values of the six peptides at 222 nm range from -31,600 to -7400 deg cm2 dmol-1 in the order listed. Changing the pH of each peptide solution from 2 to 13 also generates a nested set of dichroic spectra with the same isodichroic values. Increasing the pH from 2 to 7 differentially increases the ellipticity at 222 nm in a single transition having an apparent pK of 4.1 for the E-containing peptides are 3.6 for the D-containing peptides. Increasing the pH beyond neutrality differentially decreases the ellipticity at 222 nm in a single transition having an apparent pK of greater than or equal to 13.2 for the R-containing peptides, 11.1 for the K-containing peptides, and 10.7 for the O-containing peptides. It is proposed that the difference in the ellipticity of the six peptides chiefly reflects the helix preferences for the variable residues supplemented by intrahelical electrostatic interactions in the neutral pH range.

Dissociation of the stimulatory activities of staphylococcal enterotoxins for T cells and monocytes.

The staphylococcal enterotoxins (SEs) are homologous proteins related in their capacity for stimulating both T cells and monocytes. To assess the importance of conserved structure and sequence to functional activity, the role of the disulfide loop and adjacent sequence in these toxins was evaluated. Contrary to previous reports, we demonstrate here that the disulfide loop was required for the mitogenic activity of SEA and SEB. While T cell-stimulatory activity was compromised, reduced and alkylated SEs retained major histocompatibility complex class II-binding and monocyte-stimulatory activities, suggesting that their inability to induce T cell proliferation was due to failure to interact with T cell receptor (TCR) rather than with class II molecules. Reduction and alkylation did not affect the far-ultraviolet circular dichroic spectrum of SEA, suggesting that the loss of mitogenic activity was not associated with significant changes in secondary structure. The disulfide linkage imparts considerable stability to these toxins as peptide cleavages within the loop of SEB were not associated with detectable loss of function, although cleavage in the conserved sequence outside the loop of SEA resulted in loss of mitogenic activity. This report thus establishes a functional role for a conserved element in SEs, the disulfide loop, and further indicates that their class II- and TCR-binding activities can be dissociated.

Binding of chlorogenic acid by the isolated polyphenol-free 11 S protein of sunflower (Helianthus annuus) seed

By use of a published method for the isolation of the 11S protein of sunflower seed in a homogeneous form, a procedure has been outlined for obtaining polyphenol-free 11S protein. Neither the gel filtration nor the PAGE pattern of the protein was affected by the method of polyphenol removal. The near-and farultraviolet circular dichroism spectrum of the protein was nearly the same as that of the protein containing polyphenols. Binding of chlorogenic acid by the polyphenol-free 11S protein of sunflower seed has been measured as a function of pH, salt concentration, and temperature. Increase in pH or salt concentration decreased the binding. Binding at 45 °C was less than at 30 °C; it was completely abolished at 55 °C. Addition of Na2S03 (0.01 M), dioxane (4%), or urea (8 M) to the buffer abolished the binding. Analysis of the binding data by Scatchard equation and Hill equation showed that binding affinity was not affected by pH or salt, but the maximum number of binding sites was reduced. Decreasing the pH dissociated the 11S protein to lower molecular weight proteins. This effect was reversed by the addition of NaCl or Na2S03.

Conformational changes in human fibrinogen after in vitro phosphorylation and their relation to fibrinogen behaviour

The far-ultraviolet circular dichroism spectra of fibrinogens phosphorylated by protein kinase C or casein kinase II indicated a conformational change corresponding to an increase in ordered secondary structure. The spectra of protein kinase A- or casein kinase I-phosphorylated fibrinogens did not differ substantially from the control. Fluorescence studies indicated changes in the tertiary structure around tryptophan residues for protein kinase A- or C-phosphorylated fibrinogens, but failed to show any such change for fibrinogen phosphorylated by either of the casein kinases. This latter result was also confirmed by circular dichroism measurements in the near-ultraviolet region. The apparent increase in ordered structure was proposed as an explanation for the slower rate of plasmin degradation seen in fibrinogens after phosphorylation by protein kinase C [6], and casein kinase II, especially as both spectral changes and plasmin degradation rate were unaffected by alkaline phosphatase.

Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction

Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)

Structural organization of flagellin

The terminal regions of flagellin from Salmonella typhimurium have been reported to be disordered in solution, whereas the central part of the molecule contains protease-resistant, compact structural units. Here, conformational properties of flagellin and its proteolytic fragments were investigated and compared to characterize the domain organization and secondary structure of flagellin. Deconvolution analysis of the calorimetric melting profiles of flagellin and its fragments suggests that flagellin is composed of three co-operative units or domains. The central part of the molecule, residues 179 to 418, consists of two domains (G1 and G2), whereas the third domain (G3) is discontinuous, constructed from segments 67 to 178 and 419 to 448. Secondary structure prediction and analysis of far-ultraviolet circular dichroic spectra have revealed that G1 and G2 consist predominantly of beta-structure with a little alpha-helical content. G3 contains almost equal amounts of alpha and beta-structure, while in the terminal parts of flagellin the ordered secondary structure seems to be entirely alpha-helical.

Molecular structure of tetanus neurotoxin as revealed by Fourier transform infrared and circular dichroic spectroscopy

Secondary structure contents of tetanus neurotoxin have been estimated at neutral and acidic pH using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. An analysis of the far-ultraviolet CD spectra of the neurotoxin dissolved in 50 mM citrate-phosphate buffer (pH 7.0) revealed 20.0 +/- 2.1% alpha-helix, 50.5 +/- 2.1% beta-pleated sheets, no beta-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxin's secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the alpha-helix content estimated in this study is consistent with the earlier studies of Robinson et al. (J.P. Robinson, L.A. Holladay, J.B. Picklesimer and D. Puett, Mol. Cell. Biochem. 5 (1974) 147; J.P. Robinson, J.B. Picklesimer and D. Puett, J. Biol. Chem. 250 (1975) 7435) and with the study by Lazarovici et al. (P. Lazarovici, P. Yanai and E. Yavin, J. Biol. Chem. 262 (1986) 2645), although other secondary structural features do not agree with those of the previous studies. Secondary structure estimation from Fourier transform infrared spectra in both amide I and amide III frequency regions revealed 22-23% alpha-helix, 49-51% beta-pleated sheets and 27-28% random coils, indicating a good correlation with the secondary structure content estimated from CD analysis. Lowering of the pH of the neurotoxin to 5.5 or 4.0 did not result in any noticeable change in the overall secondary structures. However, there were significant pH-induced variations observed in the individual curve-fitted FT-IR bands in the amide III frequency region. For example, the 1302 cm-1 band (relative area, 4.2%) observed at pH 7.0 was shifted to 1297 cm-1 (relative area, 2.2%) at pH 5.5, and the relative area of the band at 1316-1317 cm-1 (alpha-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a beta-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.

Mn2(+)-binding properties of a recombinant protein-tyrosine kinase derived from the human insulin receptor.

The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn2+ in the presence or absence of 5 mM Mg2+. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn2+. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn2+ tightly in the absence or presence of MgATP (Kd greater than 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn2+, but not Mg2+, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in alpha-helical content. The addition of ATP to Mn2(+)-bound protein does not further perturb the spectrum. We conclude that Mn2+ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn2+ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin.

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.

On the Origin of the Positive Band in the Far-ultraviolet Circular Dichroic Spectrum of Fibronectin

The genesis of the positive bands in the far-ultraviolet circular dichroic spectra of human plasma fibronectin and its 31-kDa NH2-terminal heparin-binding fragment was studied. Spectra of ester derivatives of tyrosine, tryptophan, and phenylalanine and of model mixtures of these derivatives in which they are present in the same ratios as in the proteins indicate that all the aromatic side chains make substantial contributions to composite positive bands with maxima several nanometers below those of the proteins. In the presence of solvent perturbants such as polyethylene glycol and ethylene glycol, the bands of the model mixtures are red-shifted to the approximate positions they have in the spectra of the proteins. No additional red shift is seen with solvent perturbation of the proteins, suggesting that the conditions leading to this effect are satisfied within the proteins. A separate effect of solvent perturbation, an increase in amplitude of the positive band, occurs equally in solutions of free aromatic amino acid derivatives and in the proteins. This effect is used to estimate the relative accessibility of the chromophores of fibronectin and its fragment to different perturbants. The possible influence of protein secondary structure on the amplitude of the positive circular dichroic band is discussed.

Analysis of peptides for helical prediction.

Two terminally blocked peptides, acetylAETAAAKFLRQHMamide and acetylAETSSSRYLRQHMamide, were obtained by solid-phase synthesis, purified by reversed-phase chromatography, and characterized by fast atom bombardment mass spectrometry. Both peptides were soluble in aqueous solutions and remained monomeric over the concentration range examined. Changes in the temperature, pH, and trifluoroethanol concentration of solutions of each peptide produced changes in the far-ultraviolet circular dichroic spectrum characteristic of a two-state helix/coil transition. The limiting mean residue ellipticity of the coil and helix form of each peptide was estimated by addition of the denaturant guanidinium chloride at elevated temperature and by addition of trifluoroethanol at subzero temperatures, respectively. The midpoint for the thermal transition of the peptide SSSRY is lowered by about 30 degrees C relative to that of peptide AAAKF, in qualitative agreement from predictions based on helix probabilities of amino acid residues. The magnitude of the change observed in the midpoint of the thermal transitions suggests that the effect of single amino acid replacements on helix formation should be experimentally measurable.

Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin.

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% beta-sheets, 20% beta-turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IX alpha alpha and factor IX alpha beta. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% beta-structures, 22% beta-turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% beta-structures, 24% beta-turns, and 29% coils. None of these values is substantially altered as a result of increase of the pH from 7.4 to 10.5, or upon addition of Ca2+ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their gamma-carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the above pH adjustment and Ca2+ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the above pH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by beta-sheets and beta-turns. Their overall secondary structures are not influenced by Ca2+ binding and are stable to alkaline pH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the gamma-carboxyglutamic acid regions.

Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow

A new technique, continuous recycled flow (CRF) spectroscopy, has been developed for observing intermediates of any thermally induced, reversible reaction with a half-life of 10 s or longer. The structure can be probed by any spectroscopic method which does not perturb the system. Prolonged signal acquisitions of 8 h for ribonuclease A are possible. CRF was used to investigate the structure of the slow-folding intermediates of chemically intact ribonuclease A (RNase A) during thermal unfolding/folding under acidic conditions. The following conclusions were reached on the basis of the proton nuclear magnetic resonance and far-ultraviolet circular dichroism spectra of a folding intermediate(s): (A) The conformation of the detected folding intermediate(s) is similar to that of the heat-denatured protein. There is only limited formation of new structures. (B) The N-terminal alpha-helix is partially stable under these conditions and is in rapid (less than 10 ms) equilibrium with the denatured conformation. (C) There are long-range interactions between the hydrophobic residues of the N-terminal alpha-helix and the rest of the protein. These interactions persist well above the melting point. (D) An aliphatic methyl group reports on the formation of a new structure(s) that lie(s) outside of the N-terminal region. (E) The structures detected in chemically modified, nonfolding forms of the RNase A are also present in the folding intermediate(s). There are, however, additional interactions that are unique to chemically intact RNase A.

Purification and characterization of a low molecular weight basic protein from marine turtle egg white

The egg white of marine turtle ( Caretta caretta Linn.) and one species of tortoise ( Geomyda trijuga trijuga Schariggar) contain a low molecular weight basic protein. It has been purified to homogeneity from the egg white of marine turtle and characterized in terms of its major physicochemical and chemical properties. The molecular weight of this protein calculated from gel filtration, sodium dodecyl sulfate-gel electrophoresis in the presence of urea, sedimentation-diffusion data, and amino acid composition is 4300. Its isoelectric point is at pH 11.1 and intrinsic viscosity is 0.038 dl g −1 in 0.2 m NaCl. It has a Stokes radius of 12.6 Å and a diffusion coefficient of 16.50 × 10 −7 cm 2 s −1. Analysis of the far-ultraviolet circular dichroic spectrum has shown that the basic protein contains 27% β-pleated sheet and little or no α-helix. It possesses a single polypeptide chain of 40 amino acid residues with three disulfide bonds. It lacks serine, methionine, phenylalanine, and carbohydrate moiety. It binds to DNA and stimulates ATPase activity due to its strong basicity. The complex of DNA-basic protein is partially resistant to the action of DNase.

Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding.

The conformational transition observed upon addition of guanidine hydrochloride (Gdn-HCl) to solutions of oxidized Escherichia coli thioredoxin is dominated by a slow kinetic phase (time constant tau 1 = 300-800 s) that has features appropriate to a proline peptide isomerization. This observation has been interpreted as reflecting a folding pathway involving an obligatory isomerization of the imide peptide bond between isoleucine-75 and proline-76 [Kelley, R. F., & Stellwagen, E. (1984) Biochemistry 23, 5095-5102]; this peptide bond is known to have the cis configuration in the folded state [Eklund, H., Cambillan, C., Sjöberg, B.-M., Holmgren, A., Jörnvall, H., Höög, J.-O., & Brändén, C.-I. (1984) EMBO J. 3, 1443-1449]. We have tested this hypothesis by examining the conformational transitions of two thioredoxin mutants, trxA76 having an alanine substituted for proline-76 and trxA2 [Russel, M., & Model, P. (1983) J. Bacteriol. 154, 1064-1070] having proline-34 replaced with serine. Both mutant proteins display far-ultraviolet circular dichroic spectra similar to that of native wild-type thioredoxin. The tryptophan fluorescence emission of native trxA2 is equivalent to that of wild-type thioredoxin, while the emission intensity of native trxA76 at 350 nm is 2-fold greater. Tryptophan fluorescence and peptide ellipticity measurements indicate that the mutant proteins undergo two-state and reversible equilibrium unfolding transitions upon addition of guanidine hydrochloride (Gdn-HCl). These transitions are centered at 2.4 and 1.5 M Gdn-HCl for trxA2 and trxA76, respectively, as compared to a midpoint of 2.5 M denaturant for wild-type thioredoxin. As observed for wild-type thioredoxin, fluorescence measurements reveal monophasic unfolding kinetics for trxA2 at a variety of final denaturant concentrations. The tau for unfolding varies monotonically from 210 s in 2.4 M Gdn-HCl to 7 s for a final Gdn-HCl concentration of 3.5 M. Refolding of denatured trxA2 in 1.5 M Gdn-HCl detected by fluorescence measurements is described by three kinetic phases with time constants and fractional amplitudes (alpha) similar to those of wild-type thioredoxins. The fractional amplitude (alpha 1) of the slowest of these phases, tau 1 = 430 +/- 38 s in 2.0 M Gdn-HCl, decreases with final Gdn-HCl concentration. Multimixing experiments suggest that this phase results from an equilibration between denatured forms and has a tau of 34 s in 4 M denaturant, features previously observed for the wild-type protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of acid pH on the 11S protein of sunflower seed

Effect of acid pH on sunflower 11s protein has been monitored by the techniques of ultracentrifugation, polyacrylamide gel electrophoresis, viscosity, turbidity, difference ultraviolet spectra, fluorescence spectra, and near- and far-ultraviolet circular dichroism spectra. Ultracentrifugation and polyacrylamide gel electrophoresis suggest the dissociation of the protein up to pH -3.0 and reaggregation below this place. Viscosity, turbidity, difference spectra, fluorescence spectra, and circular dichroism spectra suggest the denaturation of the protein up to pH -3.0 and refolding of the protein molecule below this pH.

A high molecular weight protease in the cytosol of rat liver. II. Properties of the purified enzyme.

The properties of a soluble endoprotease from rat liver were studied. The enzyme was purified in a latent form. It sedimented as a single component with a sedimentation coefficient (S(0)20,w) of 19.8 S. Measurement by quasi-elastic light scattering gave a diffusion coefficient (D(0)20,w) of 2.5 X 10(-7) cm2 X s-1 and an effective hydrodynamic radius of 85 A. The enzyme had an unusually high molecular weight, estimated as 743,000 by sedimentation equilibrium and 722,000 by sedimentation velocity and diffusion measurements and as 760,000 by a recently developed low-angle laser light scattering method. Judging from electron microscopic observation and the calculated frictional and axial ratios, the enzyme molecule is disc-shaped. Analysis of the far-ultraviolet circular dichroic spectrum showed that the enzyme contains 50% alpha-helical, 25% beta-sheet, and 15% unordered structures with 10% beta-turns. The isoelectric point of the enzyme is 5.0. These properties indicate that the purified enzyme is a homogeneous molecule. In addition, the enzyme is a simple protein since it contains no measurable amounts of nucleic acid carbohydrate or lipid.