Thomas S. Seibles

Photooxidation of crystalline ribonuclease in the presence of methylene blue.

Abstract The photochemical action of methylene blue on ribonuclease resulted in a complete inactivation when 3 moles of oxygen was taken up per mole of enzyme. At this level of oxidation, the only chemical change observed was the photooxidation of 3 moles of histidine out of the total of 4. Up to 50 % inactivation, the decrease of histidine was found to be proportional to the decrease in enzymatic activity, in asmuchas the photooxidation of 0.5 mole of histidine resulted finally in a 52% inactivation. No changes in relative viscosity and optical rotation of ribonuclease could be noted up to 2.5 moles of oxygen uptake per mole of enzyme, whereas the decrease in enzymatic activity at this point amounted to 95%. Changes in ultraviolet absorption spectrum during the photooxidation of ribonuclease were in agreement with the changes observed in amino acid composition. Ribonuclease does not possess any sulfhydryl groups. Addition of reducing agents to the photoinactivated enzyme did not restore its activity. The possible significance of histidine to the catalytic activity of ribonuclease is discussed.

Photooxidation of bovine insulin sensitized by methylene blue

Abstract Photooxidation of insulin at pH 7.0 and at 10 ° was confined solely to the oxidation of the two histidine residues of the hormone. Photooxidation of insulin above 10 ° brought about an additional and progressive involvement of the tyrosine residues. The rate and extent of photooxidation of insulin is markedly influenced by the conformation of the molecule. A correlation has been established between photooxidation of the two histidine residues of insulin and its biological activity. The effect of photooxidation on the solvent perturbation difference spectra and on the rotatory dispersion parameters indicate that the observed inactivation of insulin was not due to changes in the secondary and tertiary structure of the hormone. Zinc-free insulin appears to have a somewhat more unfolded structure than Zn-insulin.

Reaction of reduced disulfide bonds in α-lactalbumin and β-lactoglobulin with acrylonitrile

Abstract Reduction of disulfide bonds of α-lactalbumin and β-lactoglobulin with β-mercaptoethanol and subsequent reaction with acrylonitrile was shown to be specific and was confined to the thiol groups, as determined by total amino acid analysis of the protein derivatives. The reaction results in the quantitative and specific conversion of the cysteinyl residues to S -cyanoethylcysteinyl groups. Upon acid hydrolysis, the S -cyanoethylcysteinyl groups were converted quantitatively to S -carboxyethylcysteine and were determined analytically as such.

Photooxidation of crystalline Clostridium botulinum type A toxin in the presence of methylene blue.

Abstract Photooxidation of crystalline botulinum type A toxin in the presence of traces of methylene blue results in a very rapid detoxification of the toxin. The combining power of the photochemically produced toxoid with the toxin antibody in vitro was not reduced as compared to the original toxin. Only a more extensive photooxidation of the toxin resulted in a moderate reduction of this property. Preliminary tests indicate that the protein detoxified by means of photooxidation is antigenic.

Studies on potato proteins

Protein patterns of four potato tuber varieties grown in the Northeastern United States were compared by electrophoresis and electrofocusing and found to be distinctly different. Molecular sizes of protein subunits of all four varieties were found to be uniform when charge differences between proteins were masked and disulfide bonds ruptured. Preliminary fractionation of Katahdin variety tuber proteins by dialysis against water yielded 25% globulin and 75% albumin. Further fractionation of the acidic proteins of the globulin fraction by density gradient isoelectric focusing at pH 4–6 separated three fractions isoelectric at pH 4.2, 4.4, and 5.3. Amino acid compositions of the three fractions were similar.ResumenModelos de proteinas de 4 variedades de tubérculo de papa cultivadas en los estados del Noroeste fueron comparados por electroforesis y electroenfoque encontrándose que eran claramente diferentes. Los tamaños moleculares de las sub-unidades protéicas, de las cuatro variedades se encontraron uniformes cuando las diferencias de cargas entre las proteinas fueron encubiertas y los enlaces disulfitos rotos. Fraccionamiento preliminar de las proteinas de tubérculos de la variedad Katahdin mediante diálisis, con agua, rindió 25% de globulina y 75% de albúmina. Un mayor fraccionamiento de proteínas acídicas de la fracción globulina por gradientes de densidad con enfoque isoeléctrico a pH 4–6 separaron 3 fracciones isoeléctricas a pH 4.2, 4.4 y 5.3. La composición de aminoácidos de las tres fracciones fueron similares.

On the structure of α-lactalbumin: I. Degradation studies with carboxypeptidase A and carboxypeptidase B

Abstract α-Lactalbumin is a single chain molecule with an N-terminal glutamic acid and a C-terminul leucine. The sequence of six amino acids at the C-terminal end of the molecule is: Ileu. Val. Tyr. Thr. Lys. Leu. COOH. α-Lactalbumin deprived of its C-terminal leucine could be crystallized. Tryptic digestion of α-lactalbumin resulted, in addition to the peptide fragments, in ihe liberation of free lysine and leucine in stoichionietric amounts. Cleavage of the disulfide bridges of α-lactalbumin did not change the molecular weight of the protein.

[21] Reduction and S-alkylation with acrylonitrile

Publisher Summary Alkylation of reduced proteins with acrylonitrile affords quantitative reaction with thiols. This chapter discusses the reduction and S-alkylation with acrylonitrile. This is because protein thiol groups formed by reduction of disulfide bonds with mercaptoethanol are effectively blocked by alkylation with acrylonitrile. The reaction is quantitative and the formation of cyanoethyl derivative is provided. The procedure of the recovery of S-cyanoethylated protein by lyophilization after dialysis is described. The acid hydrolysis and amino acid analysis is also focused. The chromatographic methods achieve separation and allow direct quantitation of S-carboxyethylcysteine. Under certain conditions of pH and length of exposure of reduced protein to aerylonitrile modification of lysine residues also occurs. The alkylation of amino groups of model compounds with acrylonitrile is a base-catalyzed nucleophilic addition, the rate of which is determined by the polar and steric characteristics of both reactants. The concentration of acrylonitrile may also be important in minimizing undesirable side reactions.

Purification of cell wall fragments by sucrose gradient centrifugation

SummaryA procedure for purification of cell wall fragments was developed. The method utilizes sucrose density gradients to efficiently remove soluble enzyme and membrane contaminants from the cell wall. Purification at each stage was monitored biochemically by the removal of cytoplasmic associated markers and ultrastructurally by thorough electron microscopic examination of the isolated cell wall fractions. Cell walls purified by the procedure were compared to those purified by the more conventional multiple washing procedure.

The pH dependent distribution of β-glucosidase activity in isolated particulate fractions

Abstract Higher levels of β-glucosidase activity were found in particulate fractions isolated from corn roots under alkaline compared to acid conditions. The greatest pH effect was observed on the crude cell wall fraction (1000× g for 10 min) where 19% of the total β-glucosidase activity was recovered at pH 7.7 compared to 3% at pH 6.0. Further analysis indicated that the particulate activity recovered in the 1000× g pellet was dissociated over a narrow range of pH. To determine if the pH effect was on the cell wall or a subcellular component found in the crude cell wall fraction, cell walls were separated from organelles and membranes by sucrose density gradient centrifugation. Most of the β-glucosidase activity in the crude cell wall fraction was not associated with the cell wall but with an unidentified component which equilibrated at 51% (w/w) sucrose.

Studies on the specificity of protaminase

Abstract The action of protaminase liberates all C-terminal arginine and lysines from the tryptic digest of α-lactalbumin. Protaminase is inert toward the chymotryptic digest of α-lactalbumin as carboxypeptidase is nonreactive to the tryptic digest of this protein. In accordance with the specificity of carboxypeptidase, the addition of this enzyme to a chymotryptic digest of α-lactalbumin effected a rapid liberation of all the C-terminal tyrosine and phenylalanine together with other amino acids. Protaminase hydrolyzes peptides with C-terminal arginine, lysine, and S-(β-aminoethyl)cysteine, and its action is confined to the liberation of these amino acids. Protaminase and carboxypeptidase-B are identical enzymes.