Studies on groundnut proteins. III. Physicochemical properties of arachin prepared by different methods

The homogeneity of arachin prepared by different methods was determined by the techniques of polyacrylamide gel electrophoresis, DEAE-cellulose chromatography, and ultracentrifugation. Arachin obtained by the method of Tombs (Biochem. J.96, 119, 1965) or Dawson (Anal. Biochem.41, 305, 1971) appeared to be homogeneous by these techniques. Total groundnut proteins, extracted in 1 m NaBr solution and subjected to double precipitation with 23% (NH₄)₂SO₄, also gave a homogeneous arachin preparation. These three homogeneous arachin preparations differed in their rate of hydrolysis by alpha-chymotrypsin, heat coagulation, and dissociation into subunits. However, SDS¹ and GuHCl denatured them to the same extent, as could be judged by the difference spectra. The phosphorus and carbohydrate content of the three preparations did not differ significantly.     

A high-pressure sample cell for circular dichroism studies.

The construction and operation of a preparative polyacrylamide-gel electrophoresis system is described. Emerging protein bands are collected by an intermittent pumping system which is based on the design of Brownstone ((1969) Anal. Biochem.27, 25–46). The original pressure-sensitive operation was, however, simplified to time-volume operation. Cooling of the gel by a central cooling finger, essentially according to Jovin et al. ((1964) Anal. Biochem.9, 351–369), has also been added. To accomodate the polyethylene tubing needed for intermittent collection of protein and also the central cooling finger, it is necessary to polymerize the gel in a mold before it is installed in the gel housing compartment of the electrophoresis cell. Gel concentrations of 5% and higher can be used in this system. Dilution of emerging protein samples by the intermittent collection system is kept to a minimum. This fact, together with simplicity of design makes it suitable for general preparative work with polyacrylamide-gel electrophoresis. Operation of the apparatus and resolution of protein bands are demonstrated by separation of bovine serum albumin polymers and thyroxine-binding proteins in human serum.     

Rapid evaluation of the initial and equilibrium parameters of CsCl and RbCl in equilibrium ultracentrifugation.

Two types of tables and graphs have been calculated. One type relates the initial density of the gradient solution to its equilibrium density at the bottom of the tube. The other type lists the function I(p) (defined by C. R. McEwen, 1967, Anal. Biochem.19, 23–29) and can be used to obtain the equilibrium density at any radius from the axis, provided the equilibrium density at one radius is known. Tables are calculated for CsCl and RbCl as gradient materials, and for Beckman Instruments rotors SW 41 and SW 65.     

Liberation of labile sulfur from ferredoxins by alkaline zinc reagent: an appraisal of the methylene blue method.

In disagreement with reported observation by Suhara and her colleagues (K. Suhara, S. Takemori, M. Katagiri, K. Wada, H. Kobayashi, and H. Matsubara, 1975, Anal. Biochem.68, 632–636) we found that more than 90% of labile sulfur was liberated from adrenodoxin within 5 min at 22°C. This rate was faster than those of spinach and clostridial ferredoxins, a result also at variance with Suhara's observation. At low temperature, the reaction was clearly biphasic, and spinach ferredoxin showed a similar profile. In the absence of zinc acetate, activation energies of the decomposition reaction of iron-sulfur center of OH^? were obtained as 39, 26, and 11 kcal/mol for adrenal, spinach, and clostridial ferredoxins, respectively. The adrenal reaction became faster as the dipole moment of the solvent increased. In the presence of 4 m urea and 1 m KCl, the rate was enhanced by approximately 26-fold, relative to the reaction without the addition of urea. In conclusion, the liberation reaction of adrenal labile sulfur with alkaline zinc reagent is fast at 22°C, indicating no need for modification of the original method (T. Kimura and K. Suzuki, 1967, J. Biol. Chem.242, 485–491; P. E. Brumly, R. W. Miller, and V. Massey, 1965, J. Biol. Chem.240, 2222–2228).     

An improved colorimetric assay for aspartate and ornithine transcarbamylases

An improved colorimetric assay for ornithine and aspartate transcarbamylase has been devised. The conventional method of L. M. Prescott and M. E. Jones (1969, Anal. Biochem.32, 408–419) for the detection of ureido compounds, has been optimized and standardized to a highly reproducible, sensitive, efficient, and inexpensive method for the assay of carbamyl aspartate or citrulline, the products of aspartate transcarbamylase and ornithine transcarbamylase, respectively.     

Modification of the tritium-release assays for prolyl and lysyl hydroxylases using Dowex-50 columns.

The tritium release assay of Hutton et al. (Anal. Biochem.16, 384, 1966) for prolyl hydroxylase and of Miller (Anal. Biochem.45, 202, 1972) for lysyl hydroxylase have been modified. The reaction is carried out on a microscale, and tritiated water is collected after passage of the trichloroacetic acid-soluble reaction products through a small Dowex-50 (H⁺) column instead of using a vacuum distillation apparatus as described in the original procedures. When measured by the modified procedures, both the prolyl and lysyl hydroxlyase reactions showed regions of linearity with respect to enzyme concentration, time and substrate concentration and were almost completely dependent on ascorbate and α-ketoglutarate. In addition, both reactions were completely inhibited by the iron chelator, α,α′-dipyridyl. The results indicate that these assay procedures are valid means of measuring prolyl and lysyl hydroxylase activities.     

Pigeon liver malic enzyme: Involvement of an arginyl residue at the binding site for malate and its analogs

Treatment of malic enzyme with arginine-specific reagents phenylglyoxal or 2,3-butanedione results in pseudo-first-order loss of oxidative decarboxylase activity. Inactivation by phenylglyoxal is completely prevented by saturating concentrations of NADP⁺, Mn²⁺, and substrate analog hydroxymalonate. Double log plots of pseudo-first-order rate constant versus concentration yield straight lines with identical slopes of unity for both reagents, suggesting that reaction of one molecule of reagent per active site is associated with activity loss. In parallel experiments, complete inactivation is accompanied by the incorporation of four [¹⁴C]phenylglyoxal molecules, and the loss of two arginyl residues per enzyme subunit, as determined by the colorimetric method of Yamasaki et al (R. B. Yamasaki, D. A. Shimer, and R. E. Feeney (1981) Anal. Biochem., 14, 220–226). These results confirm a 2:1 ratio for the reaction between phenylglyoxal and arginine (K. Takahashi (1968) J. Biol. Chem., 243, 6171–6179) and yield a stoichiometry of two arginine residues reacted per subunit for complete inactivation, of which one is essential for enzyme activity as determined by the statistical method of Tsou (C. L. Tsou (1962) Acta Biochim. Biophys. Sinica, 2, 203–211) and the Ray and Koshland analysis (W. J. Ray and D. E. Koshland (1961) J. Biol. Chem., 236, 1973–1979). Amino acid analysis of butanedione-modified enzyme also shows loss of arginyl residues, without significant decrease in other amino acids. Modification by phenylglyoxal does not significantly affect the affinity of this enzyme for NADPH. Binding of l-malate and its dicarboxylic acid analogs oxalate and tartronate is abolished upon modification, as is binding of the monocarboxylic acid α-hydroxybutyrate. The latter result indicates binding of the C-1 carboxyl group of the substrate to an arginyl residue on the enzyme.     

A modified rapid filtration assay of glycogen synthase.

The filter paper assay of glycogen synthase according to Thomas et al.(J. A. Thomas, K. K. Schlender, and J. Larner, 1968, Anal. Biochem.25, 486–489) has been modified to obtain results in a significantly shorter period of time with increased sensitivity and no loss in accuracy. The modified method is based on filtration on glass-fiber filter disks using a multiple filtration apparatus. The assay was examined on glycogen synthase activity present in muscle extracts and was found to be superior to the original assay procedure as regards reproducibility and time required for processing samples. The new method has been used in our laboratory for over 6 months.     

Determination of membrane-bound chloroplast RNA by the ethidium bromide fluorometric method

The method of El-Hamalawi et al. [(1975) Anal. Biochem.67, 384–391] for the fluorometric determination of nucleic acids with ethidium bromide has been adapted for the assay of membrane-associated chloroplast RNA. Membranes are stripped of RNA by incubation in a high-salt buffer lacking Mg²⁺, and the RNA is collected by magnesium phosphate-ethanol coprecipitation. RNA levels are determined by measuring the degree of enhancement of ethidium bromide fluorescence.     

Does copper-d-penicillamine catalyze the dismutation of O2−?

It has been reported (M. Younes and U. Weser, 1977, Biochem. Biophys. Res. Commun.78, 1247–1253; E. Lengfelder and E. F. Elstner, 1978, Hoppe-Seyler's Z. Physiol. Chem.359, 751–757) that the complex [Cu(I)₈Cu(II)₆(D-penicillamine)₁₂Cl]^5?-efficiently catalyzes the dismutation of O₂^? and that this activity is resistant to both EDTA and CN^?. However, careful study has demonstrated that this complex is unable to catalyze the dismutation of O₂^?, but that it slowly decomposes to simpler copper complexes which are active. Moreover, the activity which is observed is suppressed by EDTA or by Chelex 100 treatment.     

Disruption of the tryptophan binding site in the human serum albumin dimer

Monomer and dimer fractions of human serum albumin (HSA) obtained from charcoal-treated Fraction V HSA have very similar fluorescence and circular dichroism spectra, but the dimer neither binds l-tryptophan nor reacts rapidly with p-nitrophenyl acetate. The latter reaction presumably occurs in a major binding site of the monomer as many strongly bound ligands including l-tryptophan, small fatty acid anions (e.g., S.-W. M. Koh and G. E. Means, 1979, Arch. Biochem. Biophys.192, 73–79), and several drugs (e.g, N. P. Sollenne and G. E. Means, 1979, Mol. Pharmacol.15, 754–757) all decrease rates of reaction in direct proportion to their concentration. Binding data for those ligands indicate the presence of less than one binding site per molecule of charcoal-treated Fraction V HSA, and thus appear to reflect only its content of albumin monomer. The absence of that binding site in the dimer may reflect its inclusion within the dimer interface.     

Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein

The Coomassie brilliant blue G assay for proteins described by Bradford (1976) (Anal. Biochem.72, 248) was reexamined. It was found that the extinction coefficient of the dye-protein complex solution remained constant over the protein concentration range of 0.8 to 10 μg/ml of solution. This unchanging extinction coefficient, $\text{A}{}_{595}\text{=}\text{0.60 ± 0.01}\text{10}\text{μ}\text{g}$ of protein/ml of solution, enhances both the sensitivity and versatility of the assay. Selection of a volume of dye-reagent (0.5 to 5.0 ml) which dilutes the protein sample to a final concentration of 0.8 to 10 μg/ml permits the application of Beer's Law for accurate determinations of ≤0.5 to 50 μg of protein. A combination of Bradford's study and the present one indicates that most common laboratory reagents and chemicals exert little or no influence on the A₅₉₅ of the dye-reagent.     

A sensitive continuous and discontinuous photometric determination of oxygen, carbon dioxide, and carbon monoxide in gases and fluids.

The paper describes a sensitive, rapid, and precise photometric method for the continuous and discontinuous determination of O₂, CO₂, and CO. The method is based on highly specific color reactions: O₂ is determined by its reaction with alkaline catechol + Fe²⁺ yielding intensively colored products, CO₂ is determined by its color reaction with a solution of fuchsin + hydrazine; and CO is determined by its reaction with hemoglobin. The basic experimental equipment is that of the AutoAnalyzer (cf.Wolf, Zander, and Lang, 1976, Anal. Biochem.74, 585), with an additional chamber for the injection of small gas samples in the case of the discontinuous analysis. Continuously analyzing in a standardized gas flow of 1 ml · min^?1 (STPD), the lower limits of the sensitivities are 50 ppm for O₂, 100 ppm for CO₂, and 50 ppm for CO. The discontinuous analysis of the three gases requires the basic experimental equipment plus an airtight chamber. The lower limits of the amounts are 0.1 μl (STPD) for O₂, 0.2 μl for CO₂, and 0.1 μl for CO.     

Synthesis of Morin–Zinc(II) Complex and its Interaction with Serum Albumin

A morin–zinc(II) complex (MZ) was synthesized and its interaction with bovine serum albumin (BSA) were studied by molecular spectroscopy including fluorescence emission spectra, UV-visible spectra, circular dichroism (CD) spectra, three-dimensional fluorescence spectra, and synchronous fluorescence spectra. The interaction mechanism of BSA and MZ was discussed by fluorescence quenching method and Förster non-radiation energy transfer theory. The thermodynamic parameters ΔH ^θ, ΔG ^θ, ΔS ^θ at different temperatures were calculated and the results indicate the interaction is an exothermic as well as entropy-driven process. Hydrogen bond forces played the most important role in the reaction. The fluorescence probe experiment showed that the binding site of MZ is in subdomain IIA of BSA and the distance between BSA and MZ is 3.17 nm at normal body temperature. The conformation changes of BSA in presence of MZ were investigated by CD spectra and three-dimensional fluorescence spectra.     

Polypeptide composition of acetylcholine receptor purified from teleost and elasmobranch electroplax membranes

Acetylcholine receptor (AcChR) was solubilized and purified from membranes derived from electric organs of the marine fish Torpedo marmorata, Torpedo nobiliana, Narcine brasliensis, and of the freshwater eel, Electrophorus electricus, using techniques originally developed for Torpedo californica (27., 28.Biochem. Biophys. Res. Commun.49, 572–578; 1973, Biochemistry12, 852–856. The conditions used were identical in each case and the goal was to determine the degree of similarity between receptors from each source since conflicting reports have appeared with regard to polypeptide composition. The Torpedo and Narcine preparations were of high specific activity and exhibited four polypeptide components of apparent molecular weights 64, 59, 50, and 40 × 10³ upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Two components were observed upon gel electrophoresis in sodium cholate or upon sucrose density gradient centrifugation, representing monomeric and dimeric forms. Eel acetylcholine receptor exhibited three major subunits of apparent molecular weights 57, 49, and 40 × 10³. The amino acid and neutral sugar composition of the purified receptor preparations have been determined. The results support the contention that the receptor is composed of several types of polypeptide.     

Fluorescent gels as a general technique for characterizing bacterial c-type cytochromes

The technique described by Katan (Anal. Biochem. 74, 1976, 132–137) for detecting c-type cytochromes on dodecyl sulfate/polyacrylamide gels by their red fluorescence has been adapted for use with bacterial extracts. No purification is required, except for organic solvent treatment to remove lipids. A wide range of c-type cytochromes has been found to give very similar red fluorescence. Molecular weights can be estimated by means of dansylated marker proteins fluorescing green.     

几种变性剂对无花果蛋白酶分子去折叠与活力的影响

用不同浓度的变性剂盐酸胍、脲、十二烷基硫酸锂（ＬＤＳ）对无花果蛋白酶（Ｆｉｃｉｎ）变性,用荧光光谱及圆二色谱（ＣＤ谱）监测无花果蛋白酶去折叠过程中的构象变化并与活力变化比较,发现在１－２ｍｏｌ／Ｌ胍浓度及９．２×１０－４ｍｏｌ／ＬＬＤＳ浓度条件下,ＣＤ谱显示的二级结构含量较高,荧光谱的发射峰位刚开始红移,活力的变化则较为显著,表现为胍溶液中激活,ＬＤＳ溶液中失活,揭示酶的这二种变性剂的这二个浓度范围内,可能存在变性中间态。     

Study on the interaction between a water-soluble dinuclear nickel complex and bovine serum albumin by spectroscopic techniques

The interaction of a water-soluble dinuclear nickel(II) complex, [Ni₂(EGTB)(CH₃CN)₄](ClO₄)₄·4H₂O (EGTB = ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetrakis(2-benzimidazoyl)) (1), and bovine serum albumin (BSA) was investigated under physiological conditions using fluorescence, synchronous fluorescence, UV–vis absorption and circular dichroism (CD). The experimental results suggested that the nickel(II) complex could bind to BSA with binding constant (K) ~ 10⁴ M^?1 and quench the intrinsic fluorescence of BSA through a static quenching mechanism. The thermodynamic parameters, ΔG°, ΔH°, and ΔS°, calculated at different temperatures, indicated that the binding reaction was spontaneous and electrostatic interactions played a major role in this association. Based on the number of binding sites, it was considered that one molecule of complex 1 could bind to a single site or two sites of the BSA molecule or the two binding modes coexisted. In view of the results of site marker competition experiments, the reactive sites of BSA to complex 1 mainly located in subdomain IIA (site I) and subdomain IIIA (site II) of BSA. Moreover, the binding distance, r, between donor (BSA) and acceptor (complex 1) was 5.13 nm according to Förster nonradiation energy transfer theory. Finally, as shown by the UV–vis absorption, synchronous fluorescence and CD, complex 1 could induce conformation and microenvironmental changes of BSA. The results obtained herein will be of biological significance in toxicology investigation and anticancer metallodrug design.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.