Determination Of Amino Acid Composition Research Articles

The phenoloxidases of the ascomycete Podospora anserina. Structural differences between laccases of high and low molecular weight.

In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.

A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin.

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.

Tryptophanase from Sphaerophorus funduliformis. Purification, molecular weight and subunit properties

A crystalline tryptophanase can be obtained from extracts of Spaerophorus funduliformis using a heat treatment, hydroxyapatite chromatography and solubility in solutions of (NH 4) 2SO 4 as a function of pH and temperature. The purified enzyme is homogeneous by several criteria. S. funduliformis tryptophanase has a specific activity of 11.5–13.5 and requires pyridoxal 5′-phosphate for enzymatic activity. Like other tryptophanases that have been studied, the S. funduliformis enzyme is a tetramer protein consisting of four apparently identical subunits. The native enzyme has a sedimentation coefficient of 11.2 S and a molecular weight of 244 000. In solutions of 5 M guanidine · HCl, 8 M urea, and sodium dodecylsulfate, at high pH or in the presence of thiols, the enzyme dissociates to 59 000 molecular weight species which are homogeneous by the criterion of weight. Peptide maps of the reduced holo-tryptophanase show one pyridoxal-containing peptide and, lacking agreement with the determined amino acid composition, suggest that the subunits of the enzyme contain a high degree of internal sequence homology.

A new acid hydrolysis method for determining tryptophan in peptides and proteins

Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.

Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima

Phloem proteins (P-proteins) were isolated from exudates of both the fruit skin and the stem of the pumpkin, Cucurbita maxima, and the cucumber, Cucumis sativus, and were purified by ammonium sulphate precipitation and chromatography over Sephadex, DEAE-cellulose, and CM-cellulose. It was found that the fruit skin phloem is an excellent source for large-scale preparations of P-proteins which are biochemically and structurally identical with those from stem exudates. Besides fractionation on columns the P-protein preparation was characterized by electrophoresis in polyacrylamide gels (in SDS or acidic urea solutions) and on cellulose acetate, by precipitation studies using salts, acids and thiol reagents, by centrifugation techniques, by determination of amino acid composition, by cyanogen bromide cleavage, by immunodiffusion and immunoelectrophoresis using anti-P-protein-sera obtained from mice, and by electron microscopy of negatively stained and ultrathin sectioned preparations of native and reaggregated P-proteins. P-proteins were identified as a class of at least eight different but related basic (IEPs from pH 9.6 to 10.4) proteins and polypeptides of molecular weights 14000, 17000, 44000, 59000, 116000 and 158000 D which are rich in lysine, leucine, glycine, glutamic acid (plus glutamine) and aspartic acid (plus asparagine). Structurally they were pleomorphic and formed, at various proportions, floccules, fibrils, doughnuts and 100 Å lamellae with a membrane-like ultrastructure. The P-proteins showed in several properties (amino acids, IEPS, retention on CM-cellulose, antigenic sites, molecular weights of the smaller components) a strong similarity to proteins extracted from a ribosomal fraction prepared from cucurbit seedlings, in particular to a weakly basic subtraction of ribosomal proteins. It is hypothesized that P-proteins are formed during sieve element maturation by aggregation and oxidative disulphide cross-linking of preexisting proteins which, at least in the Cucurbitaceae, are basic and may include ribosomal or ribosome-associated proteins. Possible functions of these aggregates are discussed.

Immobilized Derivatives of Leucine Aminopeptidase and Aminopeptidase M: APPLICATIONS IN PROTEIN CHEMISTRY

Abstract Leucine aminopeptidase and aminopeptidase M have been covalently bound to an arylamine derivative of porous glass. The bound forms of both enzymes retain 100% of their activities at saturating levels of substrate (leucine p-nitroanilide). For the hydrolysis at pH 7.3 and 25° κcat values for bound and free leucine aminopeptidase are 46 ± 5 s-1 and 46 ± 2 s-1, respectively; for aminopeptidase M (pH 7.5 and 25°) κcat values are 23 ± 2 s-1 and 21 ± 0.4 s-1, respectively. Although the Michaelis constants for both enzymes increase on binding, the pH and temperature dependencies of the bound enzymes remain unchanged. These data suggest that the environments and conformations of the enzymes are not significantly changed after coupling to the solid support. The apparent decrease in the binding of substrate could be explained by a decrease in the effective diffusion coefficient of the substrate. Both insoluble enzymes are active against polypeptide substrates. After treatment for removal of contaminating endopeptidases, the immobilized derivatives of leucine aminopeptidase and aminopeptidase M were used successfully in NH2-terminal sequence determination. The bound aminopeptidase M appears to be the better of the two for this purpose. Both bound enzymes will catalyze the hydrolysis of the aminoethylated A and B chains of insulin nearly to completion (≥87% recovery of free amino acids in all cases). These digests are carried out at pH values near neutrality in a volatile buffer with no activating metal. Immobilized pronase (Royer, G. P., and Green, G. M. (1971) Biochem. Biophys. Res. Commun. 44, 426) was used in concert with bound leucine aminopeptidase and bound aminopeptidase M for the hydrolysis of β-lactoglobulin. In both cases the recovery of free amino acids was 93%. These bound enzymes should be quite useful in amino acid composition determinations when acid-labile residues such as tryptophan, glutamine, asparagine, or certain affinity labeled side chains are present.

Fractionation of toxins from Hydrophis cyanocinctus venom and determination of amino acid composition and end groups of Hydrophitoxin a : C.-S. Liu, G. S. Huber, C.-S. Lin and R. Q. Blackwell, Toxicon, 1973, 11, 73. (Department of Biochemistry and Toxicology Section, Department of Clinical Investigation, U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan, Republic of China and Publications Office,

Fractionation of toxins from Hydrophis cyanocinctus venom and determination of amino acid composition and end groups of Hydrophitoxin a : C.-S. Liu, G. S. Huber, C.-S. Lin and R. Q. Blackwell, Toxicon, 1973, 11, 73. (Department of Biochemistry and Toxicology Section, Department of Clinical Investigation, U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan, Republic of China and Publications Office,

Amphibian Red Blood Cell Ferritin

Erythroid ferritin was isolated from tadpole red blood cells, an unusually abundant source, with an iron content of 12%. The apoprotein was characterized by sedimentation equilibrium studies, sodium dodecyl sulfate gel electrophoresis, and by determination of amino acid composition. Tadpole red cell apoferritin has a molecular weight of 478,000, a subunit size of 19,600, and an amino acid composition similar to that of horse spleen apoferritin, particularly in its proline content. However, there is little or no cysteine in the tadpole protein in contrast to that from horse spleen; two other tadpole red cell proteins also have little or no cysteine. The amount of ferritin in amphibian red blood cells decreased from 0.9% of the soluble protein in the tadpole to 0.08% in the frog as measured immunologically; ferritin could not be detected in lysates of frog cells using gel electrophoresis. A large non-hemoglobin iron pool in the tadpole red cell was indicated not only by the amount of ferritin but also by the observation that cell suspensions could incorporate 14C-amino acids into hemoglobin without incorporating 59Fe into hemoglobin. Red cell suspensions from adult frogs incorporated 14C-amino acids and 59Fe into hemoglobin.

Fractionation of toxins from Hydrophis cyanocinctus venom and determination of amino acid composition and end groups of hydrophitoxin

Three toxic fractions were separated from crude venom of the sea snake Hydrophis cyanocinctus by column chromatography. The most abundant fraction, called Hydrophitoxin a, has been studied for amino acid composition and found to contain 19 of the common amino acids (including asparagine and glutamine); phenylalanine is absent. The total number of amino acid residues per molecule is estimated to be 59. Methionine occupies the N-terminal position and asparagine is at the C-terminal end. From these preliminary results Hydrophitoxin a appears to be similar in molecular size and structure to several other sea snake venoms previously reported.

Anthranilate Synthase from Pseudomonas putida: PURIFICATION AND PROPERTIES OF A TWO-COMPONENT ENZYME

Abstract The anthranilate synthase purified from Pseudomonas putida, is composed of 1 molecule each of two dissimilar protein components. With molecular weights of 63,400 (Component I, or AS I) and 18,000 (Component II, AS II), the pure protein components are free of chorismate mutase, and the enzymes responsible for converting anthranilate to tryptophan but retain trace l-glutaminase activities detectable only by radioisotope assay. Using the determined amino acid composition for each component, the millimolar extinction coefficients for AS I and AS II were calculated to be 5.6 and 0.9, respectively. The experimentally determined millimolar extinction coefficients were 4.4 (AS I) and 1.1 (AS II). AS I and AS II have respective specific activities of 4.7 and 17 in the conversion of chorismate to anthranilate with glutamine as nitrogen source. For this amidetransfer to occur AS II must bind AS I; high glutamine (10 mm) enhances the binding of AS I and II, as does magnesium ion, also required for the amidetransfer. The Km values for chorismate and glutamine in anthranilate synthesis at optimum pH 7.8 are 7 and 600 µm, respectively. At optimum pH 8.8, AS I by itself converts chorismate and ammonia to anthranilate at a rate greater than the maximum for the amidetransfer. The rate of this amination decreases sharply as pH is lowered to neutrality but is unaffected by the addition of AS II. With AS II present and at optimum pH the respective Km values for chorismate and ammonium ion are 0.09 and 26 mm. AS I and II play a joint role in the binding of l-glutamine since [14C]glutamine shows negligible binding to either component alone but does bind to an equimolar mixture of AS I and II with Kd = 14 µm. Additional evidence for this joint role derives from the requirement for the presence of AS I and glutamine for protection of AS II against inactivation by bromopyruvate. Glutamine analogs 6-diazo-5-oxo-l-norleucine and azaserine inactivate amidetransfer but not amination. The inactivation is delayed but not prevented by higher levels of l-glutamine. Inactivation is markedly stimulated by the presence of chorismate. Lineweaver-Burk analysis of the increased inactivation rates showed an apparent Km for chorismate of 10 µm. Although both AS I and AS II must be incubated with 6-diazo-5-oxo-l-norleucine to achieve full inactivation, resolution of 6-diazo-5-oxo-l-norleucine-inactivated anthranilate synthase into its two nonidentical components yields an active AS I and an inactive AS II. The inactivated AS II is reactivated by sulfhydryl compounds such as 2-mercaptoethanol. The importance of an —SH group on AS II placed near the active site of the native complex (AS I + AS II) is suggested.

アニサキス幼虫の食品衛生学的研究-II

Anisakiasis is said to be caused by ingestion of the excreta or body constituents of Anisakis larva. To study the chemical composition of these materials, the determination of amino acid composition was carried out. The worms werg collected from Alaska pollack caught in the Okhotsk Sea and in the Pacific Ocean. The worms were washed with physiological saline, then extracted with 70% ethanol. The residue was hydrolyzed with 6 N HC1 at 110°C for 20hrs. The determinative results with a liquid chromatography make clear the following points. Obvious differences were found between the two extractives from Okhotsk and Pacific worms as follows: in the former, large quantities of glutamic acid and alanine were found, while in the latter, the content of alanine was found to be highest. Approximately the same results were obtained from both hydrolysates, both of which contains 17 kinds of amino acids. No special ninhydrin positive substances were found. The worms collected from Alaska pollack contained 3 or 6 times the aspartic acid and glutamic acid content of worms from mackerel, but relatively lesser amounts of tyrosine, histidine and arginine.

Crystalline Milk-Clotting Protease from Mucor Miehei and Some of its Properties

A milk-clotting protease has been separated in crystalline form from filtrates of Mucor miehei. It is the major proteolytic component produced by the microorganism, and has a molecular weight of 34,000 to 39,000. Determination of amino acid composition indicates: Asp42, Thr18, Ser25, Glu15, Ala22, Val16, ½(Cys)4, Met5, Ileu11, Leu14, Tyr13, Phen14, Orn1, Lys8, His2, Arg4, Try3 (NH3)39. The pH optima for proteolysis of hemoglobin, casein and bovine serum albumin classify the enzyme as an acid protease.The enzyme is most stable at acid pH. The effects of inhibitors indicate that this clotting enzyme is not metal dependent and does not have serine or SH active groups. Aluminum sulfate has a peculiar depressing action on clotting activity, and study of the mechanism has pointed out similarities between this M. miehei enzyme and calf rennin.

Amino acid composition determination.

The pioneering studies of Emil Fischer [301] at the turn of the century established the role of amino acids and the peptide bond in the composition and chemistry of proteins. In the light of contemporary techniques, his methods of protein degradation and subsequent fractionation of the products appear cumbersome and of historical interest only. Yet it is significant that for nearly 50 years these original publications provided the major methods available to the analytical protein chemist.

Fractionation studies of the ribosomal proteins from Escherichia coli

Ribosomal proteins from Escherichia coli were fractionated by column chromatography on carboxymethyl-cellulose, using 8 m-urea solutions (pH 8.0) and a salt gradient. The resulting fractions were characterized by disc electrophoresis in polyacrylamide gels and by determination of amino acid composition and tryptic peptide distribution. Ribosomal proteins were fractionated into four classes of chemically different proteins. All but one class appeared to be complex and composed of multiple proteins by every criterion employed. An acidic protein, called ribosomal A protein, was isolated and partially characterized. On the average, each 70 s ribosome is estimated to contain two to three molecules of this protein. The chemical heterogeneity of ribosomal proteins was found to be large. Variability in amino acid sequence within a limited number of different classes of ribosomal proteins may contribute to the over-all complexity of these proteins.

Some Characteristic Properties of Hemoglobin C

Abstract 1. The present paper describes an apparently white family in which a few members suffered from homozygous and heterozygous hemoglobin C disease. Since the paternal great grandfather and the maternal grandfather of the patients examined were in a family relation of the first order, it is surmised that the hemoglobin C was derived from the same homozygous negroid (?) ancestor. Besides its occurrence in this family Hb-C is also found in a colored family from the same island (Curaçao). 2. Hemoglobin C shows, in addition to abnormal electrophoretic mobility, three other more or less specific properties: a. During chromatographic examination carried out with the aid of the cation exchanger Amberlite IRC 50 (XE 64), a citrate citric acid buffer at pH 6 and sodium ion concentration of 0.15, COHb-C displaces itself noticeably slower than the three other forms of hemoglobins (S, A and F). b. The solubility of the COHb-C in a phosphate buffer pH 6.5 of various concentrations is significantly less than that of COHb-S, COHb-A and COHb-F. The reduced form of hemoglobin C does not show this property; the solubility of this is practically the same as that of reduced adult hemoglobin. c. A comparison of the amino acid composition of hemoglobin C and hemoglobin A, S and F, shows that COHb-C contains a higher number of lysine and perhaps histidine residues. This increase amounts to four residues per mol. for lysine and one residue per mol. for histidine. The possible connection between the more basic character of the proteins and the abnormal behavior in the electrophoretic and chromatographic experiments is discussed. 3. The results obtained with five experimental methods (electrophoresis, alkali denaturation, chromatography, salting-out experiments, and amino acid composition determination) all seem to indicate that in the homozygous form of this disease only the abnormal Hb-C is present. None of the other three hemoglobins (A, S and F) were shown to be present in determinable amounts. 4. In addition to the electrophoretic abnormalities, the above three properties may be of importance in the characterization of this abnormal hemoglobin.