Studies on purothionin by chemical modifications. (65/78)

Purothionin from wheat flour was chemically modified by acetic or succinic anhydride under specific conditions. The complete modification of all amino groups of purothionin caused a large change in the net charge of the molecule, leading to the loss of the toxicity to mice and yeast. The sole tyrosyl residue in purothionin was nitrated by tetranitromethane at neutral pH or iodinated by the lactoperoxidase method. The nitro- and diiodo-derivatives of purothionin showed considerably reduced toxicity. Based on these modification studies we conclude that the positive charges of lysyl residues have an important role in the interaction with the negatively charged cell surface, and that the emergence of the toxicity of purothionin depends on a certain state of the tyrosyl residue.  (+info)

Chemical properties of the N-termini of human haemoglobin. (66/78)

The chemical properties, namely pK and reactivity, of the N-termini of oxyhaemoglobin and deoxyhaemoglobin toward acetic anhydride and 1-fluoro-2,4-dinitrobenzene (Dnp-F) were determined by the competitive-labelling approach [Kaplan, Stevenson & Hartley, (1971) Biochem. J. 124, 289-229; Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175]. At physiological pH and temperature, the valine-1 alpha and valine-1-beta amino groups had unusually low pK values, but showed only minimal changes in their pK values on deoxygenation. Between pH 7.5 and pH 8.0 a deviation was observed in the pH-reactivity profiles and the apparent pK values became markedly pH-dependent. It was found that Dnp-F, but not acetic anhydride, had an abnormally high reactivity toward the N-termini. It is concluded that the valine-1 alpha and valine-1 beta N-termini make little or no contribution to the alkaline Bohr effect at physiological pH values. The high reactivity toward Dnp-F is attributed to an interaction or binding near the N-terminal region, and the discontinuity in the pH-reactivity profile at moderate alkaline pH values to a conformational change which alters the environment of these groups.  (+info)

Facile acylation of glycerophosphocholine catalyzed by trifluoroacetic anhydride. (67/78)

A simplified procedure for the synthesis of short acyl chain phosphatidylcholines is described. A mixed fatty acid-trifluoroacetic anhydride is used to acylate sn-glycero-3-phosphocholine (GPC) which has been dissolved in trifluoroacetic acid. Yields exceeding 70% are achieved in 30-min reaction time using 1.5 to 2.0 equivalents of mixed anhydride per, GPC hydroxyl. This allows a more economical use of labeled short chain acids.  (+info)

Modifications of a macrolide antibiotic midecamycin. II. Reaction of midecamycin and 9-acetylmidecamycin with dimethylsulfoxide and acetic anhydride. (68/78)

Treatment of 9,2'-diacetylmidecamycin (2) with DMSO and acetic anhydride afforded 3''-methylthiomethyl derivative (3) preferably in the presence of pyridine. Reaction of midecamycin (1) with DMSO and acetic anhydride gave 2'-acetyl-9-dehydro-3''-methylthiomethyl derivative (9) indicating that the three hydroxyl groups reacted in a different way to the reagent. When compound 2 was reacted with DMSO and acetic anhydride in the presence of CCl4, 3''-acetoxymethyl derivative (13) was a major product, which was formed via 3 through the Pummerer rearrangement. The structures of 3, 9 and 13 were confirmed by examining NMR and mass spectra of these compounds and their deuterio analogue. They showed antimicrobial spectra similar to 1 but superior in vivo activity.  (+info)

Carboxy-terminal degradation of peptides using perfluoroacyl anhydrides. A C-terminal sequencing method. (69/78)

An accurate carboxy-terminal sequencing method has long been sought to complement the Edman degradation procedure for amino-terminal amino acid sequence analysis. The method presented here is a unique and simple method to partly fulfill the needs. Exposure of a polypeptide to perfluoroacyl anhydride vapor at -20 degrees C for 0.5-1 h causes sequential chemical degradation of the molecule from the C-terminus. Fast-atom-bombardment mass spectrometric analysis of the resultant mixture of C-terminally truncated molecules permits the determination of the C-terminal sequence by simple calculation of the mass differences in molecular ions. Experiments suggested that this C-terminal degradation proceeds by active intermediates such as oxazolone at the C-terminal carboxyl residues.  (+info)

Determination of the effective charge of a protein in solution by capillary electrophoresis. (70/78)

This paper describes two methods to estimate the effective charge of a protein in solution by capillary electrophoresis and demonstrates these methods by using representative proteins. In one method, a "charge ladder"--a series of derivatives of a protein differing by known increments of charge but differing only minimally in hydrodynamic drag--is generated by covalent modification of the epsilon-amino groups of lysines with 4-sulfophenyl isothiocyanate or acetic anhydride. In the second method, the equivalent of a charge ladder is produced by noncovalent association of a protein with differently charged ligands. Analysis of the electrophoretic mobilities of the protein and its derivatives as a function of added charge allows the effective charge to be estimated for the unmodified protein. This type of analysis permits estimation of the effective charge of a protein without knowing its composition, structure, or amino acid sequence.  (+info)

Characterization of three types of aspartase activated by site-directed mutagenesis, limited proteolysis, and acetylation. (71/78)

The activity of aspartase (L-aspartate ammonia-lyase, EC from Escherichia coli is enhanced 2- to 3-fold by three types of modification of the enzyme as reported previously; the replacement of Cys-430 with Trp by site-directed mutagenesis, the truncation of the C-terminal region by limited proteolysis, and the acetylation of amino groups with acetic anhydride. To elucidate the molecular basis of such activation, we have compared the kinetic properties of the modified enzymes in this study. Although the modifications caused very similar changes in the kinetic properties, such as increase in kcat, the half-saturating concentration of substrate, and Hill coefficient values, the modified enzymes differed greatly in sensitivity to the activator L-aspartate and the inhibitor Cl- ions. As a result of the mutation, the binding affinity for the activator was greatly decreased without change in the sensitivity to the inhibitor, whereas after acetylation, the sensitivity to the inhibitor was completely lost without decrease in the binding affinity for the activator. After truncation of the C-terminal region, both a large decrease in the binding affinity for the activator and complete loss of sensitivity to the inhibitor occurred, suggesting that this type of activation is equivalent to the former two types combined.  (+info)

Breakdown of N-terminally modified peptides and an isopeptide by rumen microorganisms. (72/78)

Treatment of Trypticase peptides with acetic anhydride, succinic anhydride, or maleic anhydride inhibited their breakdown to ammonia by rumen microorganisms by an average of 89% after 12 h of incubation in vitro. All three treatments gave similar protection. Acetylation also protected dipeptides containing lysine and methionine from degradation. However, more effective protection was obtained by linking lysine and methionine as N-epsilon-methionyl lysine.  (+info)