Leopold Weil

Photoöxidation of amino acids in the presence of methylene blue

Abstract A systematic study was made of the photochemical action of methylene blue on amino acids. Tyrosine, tryptophan, histidine, methionine, and cystine were highly reactive during the photooxidation; the rest of the amino acids acted sluggishly or not at all. In tyrosine, tryptophan, and histidine, the entire oxygen uptake and CO 2 evolution were due to the cyclic nucleus, involving rupture of the rings. During the photochemical action of methylene blue on tyrosine, tryptophan, and methionine, intermediary oxidizing agents were formed; in methionine this was shown to be H 2 O 2 . The photooxidation of methionine resulted in the formation of methionine sulfoxide as an end product. Iodometric titration and measurement of ultraviolet absorption during irradiation of methionine indicate the formation of an intermediary dehydrogenation product which appears to differ from Lavines dehydromethionine. Cystine was photooxidized, probably beyond the cysteic acid stage. Peptide bonds did not participate in the photochemical action of methylene blue. Methylation of the α-amino group of lysine to the corresponding secondary and tertiary compounds produced increased reactivity in the photooxidation.

On the mechanism of the photo-oxidation of amino acids sensitized by methylene blue

Abstract Photo-oxidation of histidine, methionine, tryptophan, and tyrosine, and their derivatives, as the function of pH were investigated. Photo-oxidation of histidine and methionine as a function of temperature has shown that oxidation proceeds through an initial fast reaction, which is followed by an additional light-sensitive slow reaction, the latter being more pronounced with increasing temperature. Photo-oxidation of tyrosine and tryptophan have also shown a marked temperature dependence. It was proposed that the first step in the photo-oxidation of histidine, methionine, tryptophan, and tyrosine consists of oxidation of these amino acids with a simultaneous reduction of the dye. Photo-oxidation of the reactive amino acids obey the Michaelis-Menten reaction scheme. A possible cyclic free radical mechanism for the photo-oxidation of amino acids mediated by methylene blue is proposed.

Photooxidation of crystalline chymotrypsin in the presence of methylene blue

Abstract Photooxidation of chymotrypsin with visible light in the presence of traces of methylene blue resulted in rapid inactivation of the enzyme. Both the proteolytic and esterolytic activities were inactivated at the same rate. A 100 % inactivation of the enzyme took place at 4.0 moles of oxygen uptake per mole of enzyme, at which point 1 mole of histidine, of the total of 2 moles, and 3 moles of tryptophan, of the total of 7 moles, were photooxidized. Solubility and viscosity measurements indicated the absence of denaturation during the photooxidation of the enzyme. No hydrolysis of the peptide bonds of chymotrypsin could be observed during the photooxidation of this enzyme. Photooxidation of chymotrypsinogen to the extent of 4 moles of oxygen per mole of the precursor eliminated its ability to be converted into the active enzyme by traces of trypsin. Photo-inactivation of chymotrypsin eliminated the reactive site for the attachement of diisopropyl fluorophosphate. The possible significance of these findings in relation to the catalytic property of chymotrypsin is discussed.

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.

Photoöxidation of crystalline lysozyme in the presence of methylene blue and its relation to enzymatic activity.

Abstract Optimal conditions for the photooxidation of lysozyme were investigated. Photooxidation of the lysozyme molecule to the extent of 2 moles of oxygen resulted in 70% reduction in enzymatic activity, in complete elimination of histidine (1 mole), and in loss of 1.2 moles tryptophan out of the total of 8 moles. Photooxidation carried out to 6 moles of oxygen per mole of enzyme eliminated the activity completely, and caused an additional loss of about 3 moles of tryptophan and about a half mole of tyrosine of the total of 3 moles. Changes in the ultraviolet absorption spectrum of lysozyme during photooxidation are presented. The observed decrease in solubility and increase in viscosity during photooxidation of lysozyme did not appear to be related to enzyme inactivation. The possible roles of tryptophan and histidine in lysozyme activity are discussed.

Photoöxidation of crystalline β-lactoglobulin in the presence of methylene blue

Summary Optimal conditions for the photochemical action of methylene blue on β-lactoglobulin were studied. It was shown that the first points of attack during photooxidation of β-lactoglobulin are the aromatic nuclei of the histidine and tryptophan residues. Tyrosine was photooxidized only after the complete oxidation of histidine and tryptophan took place in the protein molecule. A gradual decrease in the cystine content of the protein occurred during photooxidation, but methionine was not affected. A decrease in solubility and an increase in viscosity appeared to be associated with the decrease of the tryptophan content of β-lactoglobulin during photooxidation. At 20 moles oxygen uptake/mole β-lactoglobulin, the molecular weight increased to 147,000. Photochemical action of methylene blue on β-lactoglobulin did not involve hydrolysis of the peptide bonds. Photochemical action of methylene blue on lysozyme, insulin, salmine, and gelatin was studied.

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.

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.