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PubMed1976

[Cathepsin D from horse spleen. I. Purification and study of certain physicochemical properties].

Horse spleen cathepsin D (3.4.23.5.) was purified from crude extract by sodium chloride and ethanol precipitation, column chromatography fractionation on DEAE cellulose and CM Sephadex, re-chromatography on DEAE cellulose and gel filtration. The enzyme has been purified about 3.000 folds with a yield of 30 per cent. The purified enzyme seems to be homogeneous on Sephadex G100, one protein band is apparent on disc electrophoresis. Determined by dansylation the N-terminal amino acid is glycine. A molecular weight of 42,500 +/- 3,000 was obtained with Sephadex G100 gel filtration and light scattering measurements. Amino acid analysis and chemical determinations were performed: cathepsin D is a glycoprotein (2 or 3 osamine residues) including 344 amino acids and 4 disulfide bonds. Spectrophotometric data show that E1cm/1 mg/ml = 1.01 at lambda = 280 nm. ORD measurements indicate about 20 per cent of helicoidal content in the molecule.

Amino Acids↗
PubMed1990

Spectropolarimetric analysis of the core histone octamer and its subunits.

The secondary structure of the calf thymus core histone octamer, (H2A-H2B-H3-H4)2, and its two physiological subunits, the H2A-H2B dimer and (H3-H4)2 tetramer, was analyzed by ORD spectropolarimetry as a function of temperature and solvent ionic strength within the ranges of these experimental parameters where assembly of the core histone octamer exhibits pronounced sensitivity. While the secondary structure of the dimer is relatively stable from 0.1 to 2.0 M NaCl, the secondary structure of the tetramer exhibits complex changes over this range of NaCl concentrations. Both complexes exhibit only modest responses to temperature changes. ORD spectra of very high and very low concentrations of stoichiometric mixtures of the core histones revealed no evidence of changes in the ordered structure of the histones as a result of the octamer assembly process at NaCl concentrations above 0.67 M, nor were time-dependent changes detected in the secondary structure of tetramer dissolved in low ionic strength solvent. The secondary structure of the chicken erythrocyte octamer dissolved in high concentrations of ammonium sulfate, including those of our crystallization conditions, was found to be essentially unchanged from that in 2 M NaCl when examined by both ORD and CD spectropolarimetry. The two well-defined cleaved products of the H2A-H2B dimer, cH2A-H2B and cH2A-cH2B, exhibited reduced amounts of ordered structure; in the case of the doubly cleaved moiety cH2A-cH2B, the reductions were so pronounced as to suggest marked structural rearrangements.

Ammonium Sulfate↗
PubMed1978

Circular dichroism and magnetic circular dichroism of iron-sulfur proteins.

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for the 2-Fe ferredoxins from Pseudomonas putida and Spirulina maxima, Chromatium HIPIP, the 4-Fe ferredoxin from Bacillus stearothermophilus, and the 8-Fe ferredoxin from Clostridium pasteurianum. The spectral range spans the near-infrared, visible, and near ultraviolet. In all cases except oxidized 2-Fe ferredoxins, electronic absorption is observed continuously from less than 5000 cm-1 to above 30,000 cm-1. The CD spectra of the two 2-Fe ferredoxins are similar. In contrast, the CD of the 4-Fe and 8-Fe proteins, for a given 4-Fe cluster oxidation level, varies considerable with protein. MCD is less sensitive to protein environment than is CD. In the 2-Fe proteins, MCD at 5 T is appreciably smaller than the CD; in the 4-Fe and 8-Fe proteins, MCD and CD are comparable in magnitude. Both CD and MCD are more highly structured than the corresponding absorption spectra. The CD and MCD spectra reported provide a broader base than heretofore available for the characterization of iron-sulfur proteins containing 2-Fe and 4-Fe clusters and for the evaluation of electronic structural models for these clusters.

Bacteria↗
PubMed1976

Dissociation and denaturation equilibria and kinetics of a homogeneous human immunoglobulin Fab fragment.

The conformational equilibria and the kinetics of the approach to equilibrium of an IgG1 myeloma (Wes) Fab fragment (SSFab) and its mildly reduced and S-carboxyamidomethylated derivative (RAFab) were studied as a function of guanidine hydrochloride concentration. The unimolecular denaturation of SSFab, the bimolecular denaturation of RAFab, and the denaturation of Wes L chain reported previously (Rowe, E. S., and Tanford, C., (1973), Biochemistry 12, 4822) are interpreted in terms of the domain structure and evaluated in terms of the thermodynamic stability of the protein and the covalent and noncovalent interactions among its subunits. The Fd-L interactions are found to be extremely strong and are maintained at concentrations of denaturant sufficient to denature the individual domains. It is shown that all of the data are consistent with a two region structure for Fab, one composed of the vL and vH domains, and the other composed of the cL and cH domains, so that there are two sites of noncovalent Fd-L interactions. One region, identified as the C region, is found to be 10(2)-10(4) times more stable than the other; this difference in stability is attributed largely to a stronger and more extensive interaction between the domains of this region. The kinetics of the approach to equilibrium are found to be extremely slow in the center of the transitions, requiring up to a week for equilibration for RAFab, and several months for SSFab. This unusual kinetic behavior is shown to be due to the strong Fd-L interaction under conditions where the monomeric domains are unstable.

Alkylation↗
PubMed1976

An experimental method correcting for absorption flattening and scattering in suspensions of absorbing particles: circular dichroism and absorption spectra of hemoglobin in situ in red blood cells.

An experimental approach to interpretation of the anomalous absorption and circular dichroism (CD) spectra of hemoglobin in situ in red blood cells is reported. Absorption flattening effects have been overcome by use of high cell concentratons in very short light path cuvettes. Differential scattering contributions to circular dichroism have been resolved using a CD instrument capable of variable detection geometry. Scattering effects have also been resolved using media of high refractive index to match that of the red blood cell. The results are in agreement with a parellel calculational analysis of red blood cell CD spectra, which predicted the relative magnitudes of the flattening and differential scattering CD contributions. An experimental absorption spectrum has been obtained for hemoglobin in the red blood cell with scattering and flattening eliminated. This quantitatively simulates the spectrum of a hemoglobin solution. The methods described should be widely applicable to conformational studies of macromolecules in their native environment.

Circular Dichroism↗
PubMed1975

Role of tyrosine residues in mitochondrial aspartate aminotransferase from beef kidney.

Mitochondrial aspartate aminotransferase from beef kidney is 50% inhibited after 2 hr treatment with 2.5 mM tetranitromethane at pH 8. Two tyrosine residues per enzyme protomer (46,000 daltons) are modified by the reagent either in the holoenzyme or in the apoenzyme. In both cases the five SH groups titratable with p-mercuribenzoate are not modified by the reagent. However, with a tetranitromethane concentration higher than 2.5 mM and 10 mM mercaptoethanol, an additional tyrosine residue is nitrated in both holo- and apoenzymes. These results are not affected by the presence in the incubation mixture of the substrates alpha-ketoglutarate and glutamate both at ten times their Km values. Mercaptoethanol does not impair the recombination of native or nitrated apoenzyme with the coenzyme and does not reduce the coenzyme moiety of native or nitrated holoenzyme, but promotes a conformational change in the nitrated holoenzyme which causes inactivation. Hydrosulfite promotes the reduction of the coenzyme moiety of native and nitro holoenzyme resulting in their inactivation, largely in the nitrated form. The recombination of the coenzyme with native or nitrated apoenzyme is not influenced by hydrosulfite.

Animals↗
PubMed1975

The effects of salts on the subunit structure and dissociation of Lumbricus terrestris hemoglobin.

The effects of the neutral salts of the Hofmeister series, NaCl, NaClO4, MgCl2, NaI, and also guanidine hydrochloride (Gdn-HCl)on the subunit organization and the state of association of Lumbricus terrestris hemoglobin were examined by light scattering molecular weight measurements. The subunit dissociation of the parent duodecameric structure of 3 x 10(6) molecular weight by various salts is similar in pattern to the sequential splitting of the associated protein to half-molecules of hexamers of 1.5 x 10(6) molecular weight, followed by further dissociation at higher reagent concentration to monomers of 250000 molecular weight. Duodecamer to hexamer dissociation is observed in 0.4 M MgCl2, 1-2 M NaCl, and 1 M Gdn-HCl, while hexamer to monomer dissociation is seen in the presence of 1 M MgCl2. All three species of duodecamers, hexamers, and monomers seem to be present in 1 M NaClO4. Further splitting of the monomers of A subunits to smaller B fragments of one-third to one-quarter molecular weight is observed in 1 M NaI solutions. Optical rotation in the peptide region and absorption measurements in the Soret region indicate the salt dissociation of Lumbricus terrestris hemoglobin is not accompanied by major changes in the folding of the subunits, except in the case of the strong protein denaturant, Gdn-HCl. Relative to the dissociation effects of the urea series of compounds reported in the preceding paper (Herskovits and Harrington, 1975), the neutral salts appear to be much more effective dissociating agents for L. terrestris hemoglobin. This suggests that polar and ionic interactions are relatively more important for the maintenance of the protein than hydrophobic interactions. This conclusion is also supported by calculations of the possible effects of binding of NaClO4, based on the Setschenow constants of the literature describing the interaction of salts with the peptide and hydrophobic alkyl group of the average amino acid found in proteins, on the standard free energy of dissociation of the duodecamer to hexamer.

Animals↗