Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrsoine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the trytophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn²⁺-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultraviolet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77° was determined upon heating the enzyme at pH 7.5 in the presence of Mn²⁺-ions for 10 min; in the presence of EDTA, Tm is shifted to 55°. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn²⁺-ion binding.     

Interaction of urea with the high molecular weight protein fraction,carmin, from safflower seed (Carthamus tinctorius)

The effect of urea on the high molecular weight protein fraction, carmin, from safflower seed Carthamus tinctorius L, was investigated by the techniques of sedimentation velocity, viscosity, fluorescence and difference spectral measurements, partial specific volume and circular dichroism. The protein underwent dissociation and denaturation simultaneously in the presence of increasing concentration of urea. The protein dissociated to a 2S component and in the process, the buried and exposed residues of tyrosines and tryptophans were perturbed. At 8 M urea concentration, the protein existed completely as a random coil; nearly 535 mol of urea were bound per mol of protein. The results indicated that the subunits of carmin appears to be held principally by noncovalent interactions and are dissociated/denaturated by increasing concentration of urea, leading to the destabilization of the native oligomer.     

Denaturation of glycinin by urea and guanidine hydrochloride

Denaturation of glycinin by urea and guanidine hydrochloride (GdnHC1) has been studied at 30° by viscosity and circular dichroism (CD) measurements. The kinetics of denaturation by 8M urea at 20, 30, 40 and 50°, and by 6M GdnHC1 at 20° have been measured by monitoring the increase in absorbance at 287 nm as a function of time. Viscosity increased with denaturant concentration and reached maximum value of 28 mL/g at 6M GdnHC1 and 7M urea. The (negative) molar ellipticity values in the region 250–200 nm decreased with increase in denaturant concentration. Analysis of viscosity and CD data indicated that both sets of data fitted the same curve of f_d (fraction of protein denatured) versus denaturant concentration. The kinetic data followed first order reaction kinetics, These suggest that denaturation of glycinin by urea/GdnHC1 is a two-state process and follows the same pattern as that of globular proteins.     

CIRCULAR DICHROISM STUDIES ON NATIVE AND PHENYLMETHANESULFONYL-MESENTERICOPEPTIDASE

At neutral pH the far ultraviolet circular dichroism (CD) spectrum of alkaline mesentericopeptidase is dominated by two negative bands: at 208 and 220 nm. The near ultraviolet CD spectrum is characterized by a large negative band at 277nm, a shoulder near 286nm and a small positive band at 297nm. The introduction of phenylmethanesulfonyl (PMS) group on the serine residue at the active site does not alter the intensity and the wavelength position of the bands. In contrast to the other alkaline bacterial proteases, which are remarkably resistant to denaturation by urea, mesentericopeptidase was unfolded in the presence of this reagent at neutral pH. The denaturation reactions in the presence of 9.5 M urea of both native and PMS-mesentericopeptidase followed first order kinetics. The introduction of PMS-group on the active-site serine lowered the conformational stability of the enzyme. In the first 3h PMS-mesentericopeptidase was denaturated 2.5 times faster than native protease. In the presence of 9.5 M urea at natural pH alkaline mesentericopeptidase lost its catalytic activity. The loss of enzymatic activity correlated with the unfolding of the protein molecule and also followed first-order kinetics.     

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.     

Domain characteristics of the cyanogen bromide fragment 121–316 of thermolysin

The molecule of thermolysin was shown by X-ray crystallography to be composed of two structural domains of equal size comprising residues 1–157 and 158–316. In order to explore the possibility that these domains correspond to globular fragments able to refold autonomously, we have investigated the conformational and stability properties of fragment 121–316, which was obtained by limited chemical cleavage of thermolysin with cyanogen bromide. As judged by far-ultraviolet circular dichroism measurements, in aqueous solution under neutral conditions the fragment maintains a relative amount of helical structure which is comparable to that exhibited by the corresponding region in native thermolysin. The secondary structure attained by the fragment appears remarkably stable to the denaturing action of heat. By measuring the temperature dependence of the dichroic signal at 220 nm a Tm near 74d? was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates antibodies raised in rabbits using native thermolysin as immunogen. The overall conformational and immunochemical data indicate that fragment 121–316 of thermolysin is able to refold into a stable structure of native-like characteristics independently of the rest of the molecule. The results of this study complement those previously reported for fragment 206–316 (Vita, C., Fontana, A., Seeman, J.R. & Chaiken, I.M. (1979) Biochemistry 18 , 3023–3031).     

Alkaline unfolding and salt-induced folding of yeast alcohol dehydrogenase under high pH conditions

The conformational changes of yeast alcohol dehydrogenase during unfolding at alkaline pH have been followed by fluorescence emission and circular dichroism spectra. A result of comparison of inactivation and conformational changes shows that much lower values of alkaline pH are required to bring about inactivation than significant conformational change of the enzyme molecule. At pH 9.5, although the enzyme has been completely inactivated, no marked conformational changes can be observed. Even at pH 12, the apparently fully unfolded enzyme retains some ordered secondary structure. After removal of Zn²⁺ from the enzyme molecule, the conformational stability decreased. At pH 12 by adding the salt, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing. Folded states induced by salt bound ANS strongly, indicating the existence of increased hydrophobic surface. More extensive studies showed that although apo-YADH and holo-YADH exhibited similar behavior, the folding cooperative ability of apo-enzyme was lower than that of holo-enzyme. The above results suggest that the zinc ion plays an important role in helping the folding of YADH and in stabilizing its native conformation. © Munksgaard 1996.     

FLUORESCENCE SPECTRA OF LYSOZYME EXCITED AT 305 NM IN PRESENCE OF UREA

Tryptophan 108 of hen egg white lysozyme was selectively excited at 305 nm and fluorescence spectra were recorded as a function of pH (2–9) and concentration of urea (0–8 M). Urea at low concentrations (1–4 M) quenches markedly the Trp 108 fluorescence around pH 7; the Λ_max, however, remains unaltered. The fluorescence quenching by urea is most likely due to local conformational changes around Trp 108 in the active site region of the enzyme. Substantial unfolding of the enzyme, however, was brought about by 4M urea below pH 3, and by 7 M urea at pH 10.3, as indicated by a marked red shift in the Λ_max of the fluorescence emission.     

COMPARATIVE STUDY OF THE HIGH MOLECULAR WEIGHT PROTEIN FRACTION OF MUSTARD (B. JUNCEA) AND RAPESEED (B. CAMPESTRIS)

A method is described for isolating the high mol. wt. protein fraction of mustard and rapeseed in a homogeneous form. The protein from the two species had nearly identical sedimentation coefficients, molecular weights, intrinsic viscosity and fluorescence emission spectrum. However, differences in the amino acid composition were observed. Data on circular dichroism and rate of hydrolysis by proteolytic enzymes suggested differences in the secondary structures and possibly amino acid sequence.     

Trypsin-pancreatic secretory isoinhibitor A from bovine pancreas (Kazal type)

The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.     

Protein analysis of enzyme tablets

Enzyme tablets with butyrylcholine esterase (CHE) and peroxidase (POD) partly lose enzymatic activity during compaction at a pressure of 495 MPa. Compared to solutions of the original enzyme, no changes of ultraviolet absorbance and fluorescence intensity in the tablet solutions were found. Only small changes were observed in the far ultraviolet circular dichroism spectra. Neither missing nor additional bands were detected with polyacrylamide gel electrophoresis. Heated (150 degrees C) solid starting material with CHE and POD showed still part of its original enzymatic activity. The ultraviolet absorbance increased with continued heating until precipitation occurred. The circular dichroism spectra are changed clearly.     

Effect of pH on poppy seed 10S protein

The effect of acid pH on the 10S protein of poppy seed has been studied by ultracentrifugation, polyacrylamide gel electrophoresis, turbidity, and difference, fluorescence and circular dichroism spectra. The protein dissociated into lower molecular weight fractions in the pH range of 4.0 down to 2.5 judged by ultracentrifugation, electrophoresis and turbidity measurements. U.v. difference spectra and fluorescence measurements suggested denaturation of the protein. Thus, both dissociation and denaturation occurred down to pH 2.5. At more acid pH values (pH 1.3), reassociation and refolding probably occurred. Circular dichroism studies showed a large increase in the amplitude of the peak at pH values of 2.8 and 1.3 indicating that unordered structures are formed at acid pH values.     

Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca (Jararaca)

— Bothropasin, one of the proteases from the venom of Bothrops jararaca active on casein, was isolated by ammonium sulfate precipitation, DEAE-cellulose and DEAE-Sephadex A-50 chromatographies and Sephadex G-100 column filtration. The preparation possessed no other detectable activities which are present in the crude venom. Addition of Ca²⁺ during purification stabilized the enzyme. The endopeptidase was inhibited by EDTA and EGTA; Ca²⁺ did not restore the activity of the inhibited enzyme. The material was homogeneous by polyacrylamide gel electrophoreses at different pH values, immunoprecipitation and crossed immunoelectrophoresis. By SDS-polyacrylamide gel electrophoresis the denatured and reduced enzyme had only a 48,000 molecular weight band. In the presence of 6 M guanidine-HCl and 0.1 M β-mercaptoethanol the preparation showed a value of 49,870 by sedimentation equilibrium. The native tertiary structure of the protein is dependent on S-S and metal bonds. The denatured and reduced enzyme, in the presence of EDTA, showed a molecular weight of 37,300 by sedimentation equilibrium, a value which was also confirmed in SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed five peptide bonds: His-Leu (5–6), His-Leu(10–11), Ala-Leu(14–15), Tyr-Leu(16–17) and Phe-Phe(24–25) in the B-chain of oxidized insulin.     

Purification and conformation of ribosomal protein L25 from E. coli ribosome

Ribosomal protein L25 from the large subunit of E. coli ribosomes has been purified using a new procedure involving a 2 m LiCl extraction followed by phosphocellulose chromatography in 6 m urea elution buffer. The conformation of purified L25 was studied employing circular dichroism and ultraviolet absorption spectroscopy in reconstitution buffer. The analysis of the far u.v. circular dichroism spectrum of L25 indicates L25 contains ～ 16%α-helix and ～ 19%β-structure. The conformation of L25 was also studied using the predictive methods of Chou & Fasman and Maxfield & Scheraga. Both of these methods predict approximately three times the percent α-helix present in L25 as compared with that determined from the analysis of the circular dichroism spectrum. A structure for L25 is predicted which contains two positively charged binding domains and is consistent with published binding data on the interaction of 5S RNA and L25. The large difference in the %α-helix as determined from the analysis of the circular dichroism spectrum and the predictive techniques is suggested to result from the denaturing effects of 6 m urea used in the preparation of ribosomal proteins.     

Conformational changes of urea-denatured colicin E1 induced by phospholipid membranes

Abstract: The membrane insertion of urea-denatured colicin E1 was studied by using fluorescence spectroscopy, circular dichroism and monolayer techniques. The results showed that the denatured colicin E1 taking mainly the ‘random coil’ conformation may recover its orderliness to a certain extent under the induction of the phospholipid membrane and insert spontaneously into phospholipid membrane, indicating that unfolding of colicin E1 does not inhibit its membrane insertion. Among the four tryptophan residues of the membrane-bound colicin E1 molecules, at least two were accessible by the quenchers, i.e. not inserted into the membranes. Although urea-denatured colicin E1 interacted preferentially with negatively charged phospohlipids, it seems less dependent on the negatively charged lipid than colicin A. The addition of urea increased the speed of the adsorption of colicin E1 to the membrane, but did not affect obviously its membrane insertion ability.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule     

Conformation and structure of mildly heat-treated ovalbumin in dilute solutions and gel formation at higher protein concentrations

The conformation of heat-denatured ovalbumin aggregates has been examined at several concentrations and pH values, using measurements of optical rotation dispersion (ORD), circular dichroism (CD) and viscosity. The protein was subjected to heating at relatively low temperatures, ranging from 48.5 to 76d?; the particular temperature chosen depended on pH. The heat treatment was sufficient to remove the ability of the molecules to absorb heat on re-heating. The denatured molecules were shown to be rather compact, i.e. not much larger than the native molecule, and to retain a significant amount of secondary structure; this was also the case for molecules present in small aggregates. It is suggested that this type of ovalbumin monomer builds three-dimensional networks in denatured solutions at higher concentrations, and that gelation should be looked upon as arising from surface contacts between hydrated globules. The present results also imply that such globules have gelation properties which depend on whether pH is acidic or basic compared to the isoelectric point of the protein.     

Preparation of clear solutions of reconstituted acetylcholinesterase Effect of ionic strength and lipid/protein ratio

The detergent-soluble globular dimer of acetylcholinesterase from Torpedo californica was reconstituted through dialysis into preformed egg phosphatidylcholine vesicles. The formation of the enzyme-lipid complexes depended on the ionic strength of the dialysis buffer as well as the molar lipid/protein ratio (R). The enzyme was unstable at I < 0.05; increasing the ionic strength increased the size of the complex. A too low R value (e.g. 1000) would promote self-aggregation of the enzyme and produce heterogeneous complexes, especially at high I values. On the other hand, a too high R value (e.g. > 5000) favored the formation of large enzyme-lipid complexes; their solutions were too turbid for optical studies. The enzyme reconstituted at I = 0.07 and R = 4000 gave a clear solution and showed no artifacts due to light scattering. The conformation based on circular dichroism and enzymatic activity of the detergent-soluble enzyme were unchanged upon reconstitution. The reconstituted enzyme in lipid vesicles seemed to be slightly more stable against thermal denaturation than the protein in sodium cholate solution.     

Structure characterization of human serum proteins in solution and dry state

Abstract: The present report describes application of advanced analytical methods to establish correlation between changes in human serum proteins of patients with coronary atherosclerosis (protein metabolism) before and after moderate beer consumption. Intrinsic fluorescence, circular dichroism (CD), differential scanning calorimetry and hydrophobicity (S_o) were used to study human serum proteins. Globulin and albumin from human serum (HSG and HSA, respectively) were denatured with 8 m urea as the maximal concentration. The results obtained provided evidence of differences in their secondary and tertiary structures. The thermal denaturation of HSA and HSG expressed in temperature of denaturation (T_d, °C), enthalpy (ΔH, kcal/mol) and entropy (ΔS kcal/mol K) showed qualitative changes in these protein fractions, which were characterized and compared with fluorescence and CD. Number of hydrogen bonds (n) ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). Unfolding of HSA and HSG fractions is a result of promoted interactions between exposed functional groups, which involve conformational changes of α‐helix, β‐sheet and aperiodic structure. Here evidence is provided that the loosening of the human serum protein structure takes place primarily in various concentrations of urea before and after beer consumption (BC). Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption. In summary, thermal denaturation parameters, fluorescence, S_o and the content of secondary structure have shown that HSG is more stable fraction than HSA.     

Secondary structure of interleukin-2(Alal25) in unfolded state*

Secondary structure of interleukin-2(Alal25) in unfolded state was examined by circular dichroism (CD). Unfolding of tertiary structure of the protein, as determined by CD, was observed when the solvent pH was decreased below 3.0 or the disulfide bond was reduced. Consistent with the CD results, a stronger fluorescence enhancement of 1-anilinonaphthalene-8-sulfonic acid was observed on acidification or reduction of interleukin-2(Alal25) relative to that of the native protein, indicating a larger hydrophobic surface exposed to solvent. However, the secondary structure was fully retained in 5% acetic acid or aqueous HCl, pH 3.0. It seemed that α-helical content of the protein is even greater at pH 2.0. Reduced protein showed a far u.v. CD spectrum indistinguishable from the oxidized one at pH 4.0. These results suggest that the secondary structure of interleukin-2(Alal25) does not require tertiary structure.