Free Α-amino Group Research Articles

Studies on acylase activity and microorganisms. XXIV. Properties of delta-ornithine acylase (5-N-acylornithine amidohydrolase).

In order to study the enzymatic properties of the δ-ornithine acylase in KT 801 (Pseudomonas sp.), experiments were carried out with the purified enzyme preparation. This enzyme has a pH optimum around 8.2 toward 5-N-benzoyl-L-ornithine. It is inhibited by Co2+, Zn2+, Fe3+, Cu2+, Hg2+, EDTA, and o-phenanthroline. The inactivation for EDTA is reversed by addition of Ca2+ or Mn2+ after dialysis. Km value toward 5-N-benzoyl-L-ornithine was calculated to be 1.8×10-4M. As a results of the investigation of substrate specificity, it was found that this enzyme hydrolyses not only L form of 5-N-benzoylornithine, but D form in the rate of about one sixth. The ratio of the activities toward L and D form remained constant all through the purification steps. These result strongly suggest that one enzyme has the both activities. Also, susceptible substrate to δ-ornithine acylase require absolutely the presence of a free carboxyl group, the presence of a free α-amino group is desirable for ready susceptibility of the substrate, but it is not an absolute requirement, and the susceptibility are considerably affected by carbon chain length between the carboxyamide group and the free carboxyl group. This enzyme shows no α-amino acylase activity. On the other hand, δ-ornithine acylase is quantitatively released when EDTA-lysozyme spheroplasts are made. From these result, it has been proposed that δ-ornithine acylase of KT 801 is at the cell surface.

Aeromonas aminopeptidase: Purification and some general properties

An aminopeptidase was purified 87-fold from culture filtrates of the marine bacterium Aeromonas proteolytica. The stability of the aminopeptidase at 70 ° permitted the use of a heat treatment to inactivate a proteinase from which the aminopeptidase was otherwise difficult to separate. Purification of the aminopeptidase from heat-treated culture filtrate was accomplished by chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. Moving-boundary electrophoresis, ultracentrifugation, and substrate specificity studies indicated a high degree of homogeneity for the preparations. The enzyme hydrolyzed a number of peptides, amino acid amides, and amino acid β-naphthylamides. It was specific for substrates having a free α-amino group on a residue of the l-configuration; the nature of the amino acid in the N-terminus exerted a considerable effect on hydrolytic rates. The aminopeptidase, as isolated, did not require the addition of activating ions, but was strongly inhibited by ethylenediaminetetraacetate (EDTA). Addition of Zn ++ or Co ++ to the EDTA-inhibited enzyme restored activity to the original level; Mn ++ was partially effective, and Mg ++, Ca ++, and Ni ++ were ineffective. Analysis of several individual preparations revealed the presence of zinc in the aminopeptidase. A molecular weight of 29,500 was calculated from measurements of sedimentation velocity and diffusion.

Thyroxine-protein interactions: II. The binding of thyroxine and its analogues to human serum albumin

The relative affinities of various thyroxine analogues for albumin have been determined. Estimations of relative binding affinities were based on a comparative study of the abilities of the analogues to displace thyroxine from binding sites on albumin. The results indicate that the substituents on thyroxine most directly involved in binding are: (1) the phenolic hydroxyl group; (2) iodine atoms in both the 3′- and 5′-positions of the thyronine molecule; (3) the diphenyl ether group. The absence of any one of these substituents from the molecule leads to an appreciable reduction in relative binding affinity. With regard to the alanine side chain of thyroxine, it appears that neither a free carboxylate ion nor a free α-amino group are required for binding. The results of experiments on the effect of d-thyroxine on the binding of l-thyroxine indicate that binding is not stereospecific. The constants for the binding of tetraiodothyropropionic acid by albumin have been determined.

N-Formyl-methionyl-S-RNA

The reaction between methionine and S-RNA of Escherichia coli has been investigated. It has been demonstrated that, after the initial attachment to its specific S-RNA, the free α-amino group of the attached methionine may become formylated.

The N-Terminal Amino-acids of α-Casein

As a result of previous work1–3, it has been concluded that there exist in α-casein, arginine and lysine residues carrying free α-amino groups and occupying N-terminal positions in open polypeptide chains. Estimates of the amounts of such arginine and lysine have however varied within wide limits. Furthermore, the α-casein employed in two at least of these investigations1,2 was prepared using a fractionation procedure now known to yield α-casein contaminated with k-casein.

The influence of pH on the antibacterial action of subtilin A.

Summary: The influence of pH value on the antibacterial action of subtilin A has been demonstrated by survivor counts, inhibition of respiration, and pH gradient plates. Staphylococcus aureus is more sensitive to subtilin as the pH value increases; Escherichia coli is more sensitive as the pH value decreases. The results are analogous to those obtained by other investigators working with cationic detergents, and are consistent with the hypothesis that the basic surface-active antibiotics kill bacteria by the same general mechanism as do the quaternary ammonium germicides. A survey of the growth inhibition of other organisms on pH gradient plates indicates that Bacillus cereus behaves like S. aureus, while B. megaterium, Corynebacterium poin- settiae, and Streptococcus faecalis behave like E. coli. A pH/mobility curve for subtilin A, obtained by paper electrophoretic studies, indicates no striking changes in the charge of the molecule over the pH range 4–9, although the presence of at least one free α-amino group is indicated by inflexion points in the range of pH 6–7. The isoelectric point of subtilin A at ionic strength 0·1 is approximately 6·7. The use of sectored, square-shaped Petri dishes for pH-gradient plate studies with germicides is described.

Étude chimique de la mucoprotéine responsable du phénomène d'obstacle de donaggio

The glycoprotein extracted from the urine of cancerous patients is shown by electrophoresis to be a mixture of substances; the active fraction can be isolated in a pure state by zone electrophoresis on starch or on ‘Hyflo-super-cel’. This fraction is rich in dicarboxylic amino acids and in glycine. The free α-amino groups belong principally to glutamic acid and aspartic acid; the free α-carboxylic groups to glycine, tryptophan and threonine. The molecular proportions of the ‘oses’ and of ‘osamine’ are as follows: galactose 10, glucose 2, mannose 7, fucose 7, osamine 11. The large proportion of fucose is characteristic, and the oses/osamine ratio is quite low (2.36), as a consequence of the high level of osamine. Donaggio's substance is thus rather distinct from the urinary glycoprotein isolated by adsorption on benzoic acid and from the glycoprotein that inhibits the hemagglutination with different viruses.

The chain weight of wool keratin

1. 1. A method is described for the separation of DNP-amino-acids, using buffered silica gel columns. 2. 2. By treatment with FDNB, and separation of the DNP-amino-acids after hydrolysis, wool is shown to contain seven different amino-acid residues having free α-amino groups i.e. valine, alanine, glycine, threonine, serine, glutamic acid and aspartic acid. 3. 3. The end-groups have been quantitatively estimated and the average chain weight determined. 4. 4. A molecular model is proposed which is in accordance with these values.

Studies of the reaction between proteins and reducing sugars in the ‘dry’ state II. Further observations on the formation of the casein-glucose complex

1. 1. The reaction of free amino groups of casein with glucose has been furher investigated by Van Slyke determinations. 1.1. a) The extent of the reaction at a water content corresponding to 20% R.H. has been found to depend on the degree of hydration of the protein surface. Most of the individual amino groups appear to have the same reactivity. 1.2. b)At 70% R.H. rate and extent of the reaction approach a maximum when three equivalents of glucose are present, this quantity approximating to that required to provide a unimolecular layer on the surface of the protein. 2. 2. 1.2.4 fluorodinitrobenzene has also been used for estimation of the free amino groups. 1. a)It has been shown that all the ϵ-amino groups of the lysine side chains (51 mg amino-N/g T.N.) are free to react with the reagent, and that a small quantity of free α-amino groups ( ca i mg amino-N/g T.N.) occur at the ends of polypeptide chains: these α-groups are derived from at least five amino acids, one of which is lysine. 1. b) The loss of amino-N during the reaction with glucose, as found by the Van Slyke method, has been quantitatively confirmed. Both the ϵ-amino groups and the α-amino groups have approximately the same reactivity towards glucose. 1. 3. The rate of disapperance and the fate of the glucose have been followed by various procedures, including use of the specific glucose oxidase (notatin). 1. a) Combination of the free amino groups of casein, each with one molecule of glucose, has been confirmed as the most rapid and virtually the only reaction occurring at 20% R.H., and, in the earliest stages at 70% R.H. As storage proceeds at the higher humidity progressively more carbohydrate becomes bound to the protein than can be accounted for by combination with free amino groups: its weight indicates an average loss of one molecule of water per glucose molecule. 2. b) The nature of the union between the extra carbohydrate and the protein is still uncertain. It may be a chemical union with reactive groups of the protein molecule other than amino groups, i.e., with amino acid residues other than those of lysine. Alternatively, but less probably, it may be the result of caramelization of the glucose followed by adsorption of the product on to the protein. 3. c) None of the carbohydrate combined with (or adsorbed by) the casein can be oxidized by notatin. 4. d) Hydrolysis with dilute acid or alkali fails to regenerate any glucose (or fructose) from the casein-glucose complex, although other reducing substances including furfurals are liberated by the acid.

Identification of the End-Amino Groups of Wool by Means of their 2 : 4-Dinitrophenyl Derivatives

THE amino-acids with free α-amino groups in wool have been identified using Dr. F. Sanger's technique1. Considerable use was made of buffered, as well as unbuffered, silica gel columns, using Sanger's original solvents. The gel was buffered at pH 6.0 with 0.5 ml. buffer solution (9.36 gm. NaH2PO4.2H2O + 14.32 gm. Na2HPO4.12H2O per 100 ml. solution) per gm. silica. This was used in conjunction with (a) ether, (b) 1 per cent butanol–chloroform, (c) 30 per cent butanol–chloroform, (d) 5 per cent propanol–eyclohexane, (e) 33 per cent ether-ligroin as solvents. The ether was freed from peroxides by shaking with an acid solution of ferrous ammonium sulphate, followed by several changes of distilled water, and stored in a dark bottle. In agreement with other workers2,3, leucine could not be separated from valine on unbuffered columns, but this was easily achieved by using 0·5 ml. solution (7·16 gm. Na2HPO4.12H2O + 7·60 gm. Na3PO4.12H2O per 100 ml. solution) per gm. silica with ether as solvent. The acids were identified as (a) valine, (b) alanine, (c) glycine, (d) threonine, (e) serine, (f) glutamic acid, (g) aspartio acid.

The Acetylation of Silk Fibroin by the Action of Ketene. Part II

1. The study of the behavior of ketene towards silk fibroin has been continued. 2. The rate of absorption of ketene by fibroin increases with rise of temperature, and with increase of the concentration of ketene exposed to the fibre. 3. That ketene combines with the phenolic group of typosine, and the free α-amino groups in fibroin is evidenced by the failure of completely ketenized silk to respond to the characteristic Millon and Ninhydrin tests of protein respectively. 4. More ketene is absorbed by fibroin than can be accounted for by addition only in the known. phenolic and free amino groups. The results of acetyl analyses of fibroin, saturated with ketene, indicate the presence of other unidentified groupings in the protein molecule capable of combining with ketene. 5. The degree of acetylation of degummed silk fibroin reaches a maximum or limiting value of about 7% of acetyl groups. 6. Exhaustive treatment of silk fibroin with ketone gas produces a tan coloration of the fibre.