Ernesto Scoffone

Dye-sensitized selective photooxidation of methioxine

Abstract The photooxidation, sensitized by rose bengal and methylene blue, of cystine, methionine, histidine, tyrosine, tryptophan and of some related peptides was studied in acid media. Rose bengal in formic acid solution, and methylene blue or rose bengal in aqueous acetic acid solution sensitized a selective oxidation of methionine, which was quantitatively converted to methionine sulphoxide. Irradiation of ribonuclease A (ribonucleate pyrimidinenucleotide-2′-transferase (cyclizing), EC 2.7.7.16) under the same conditions caused the modification of the four methionyl residues and a concomitant 87% decrease of the enzymatic activity, which was correlated to a conformational change of the protein. Chemical reduction of the photooxidized ribonuclease A by thioglycolic acid resulted in the full recovery of the enzymatic activity.

Reversible acetoacetylation of amino groups in proteins

A method is described by which amino groups of proteins such as ribonuclease (ribonucleate pyrimidinonucleotide-2′-transferase (cyclizing), EC 2.7.7.16) and lysozyme (N-acetyl-muramide glycanohydrolase, EC 3.2.1.17) can be acetoacetylated by reaction with diketene. The acetoacetyl group can be removed at 25° from the acetoacetylated enzymes by treatment with hydroxylamine hydrochloride at pH 7. The material thus obtained resembles native proteins in its properties and recovers the full enzymatic activity. The tryptic hydrolysis of acetoacetylated oxidized ribonuclease A has been studied in connection with the investigation of covalent structure in proteins. The lysyl bonds in the protein molecule are rendered resistant to hydrolysis by trypsin while the arginyl bonds are split off.

Photo-oxidation of horse heart cytochrome c. Evidence for methionine-80 as a heme ligand

In a previous paper [I] , we showed that the irradiation of a protein containing a photosensitizer covalently linked to known positions in the molecule, causes the selective modification of only those potentially photo-oxidizable amino acid residues which are in close proximity to the sensitizer. Therefore, once the modified side chains have been identified, their location in the three-dimensional network of the protein molecule can be deduced. In principle, it should be possible to extend this method to proteins naturally possessing a chromophoric group which can act as a photosensitizer. As a first approach we attempted to explore the environment of the porphyrin prosthetic group in horse heart cytochrome c. Since porphyrins efficiently mediate the photo-oxidation of amino acids [2], it was expected that the porphyrin group of this protein would likewise serve as such a photosensitizer. Cytochrome c was chosen also because its primary structure is known, and its biological, physical and chemical properties have been studied in detail [3] . The results presented in this communication show that only those susceptible amino acids which are adjacent to the heme group are photo-oxidized upon irradiation of ferricytochrome c. Furthermore, our data provide compelling evidence that Met-80 serves as a protein ligand for the F

[40] Sulfenyl halides as modifying reagents for polypeptides and proteins.

+ ion in ferricytochrome c. 2. Materials and methods

Isolation and some properties of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus☆

Publisher Summary This chapter discusses sulfenyl halides as modifying reagents for polypeptides and proteins. Sulfenyl halides have several particularly valuable features for protein modification studies. Their high selectivity for tryptophan in proteins lacking SH-groups is remarkable. Even if cysteine residues are present, selectivity for tryptophan can be achieved, since the mixed disulfides formed from SH-groups are easily cleaved by reduction with β-mercaptoethanol. Other techniques for tryptophan modification, such as reaction with N-bromosuccinimide, are less specific. The Koshland reagent, 2-hydroxy-5-nitrobenzyl bromide, upon reaction with tryptophan gives several derivatives allowing formation of different species of the labeled protein, as shown with pepsin. The sulfenylation reaction increases the number of procedures available for the modification of tryptophan, providing greater experimental latitude in the study of the biological function of tryptophan-containing enzymes. One of the main advantages of this technique is the possibility of using sulfenyl halides carrying different groups, thus leading to a change in size, polarity, or other physicochemical properties of the labeled enzymes.

Studies on cytochrome c. Part II. Synthesis of the protected heptapeptide (sequence 17–23) of Baker's yeast iso‐1‐cytochrome c

Abstract A purification procedure is described for 6-phosphogluconate dehdyrogenase (6-phospho- d -gluconate:NADP oxidoreductase (decarboxylating), EC 1.1.1.44) of Bacillus stearothermophilus (NCA 1503). A 1200-fold purification was obtained with a 15% yield. The purified enzyme has an approximate mol. wt of 101 000 as estimated by sucrose density gradient centrifugation and consists of subunits of a mol. wt of 51 000. Sulfhydryl group(s) are essential for enzymatic activity as indicated by p -chloromercuribenzoate and Ellmans reagent inactivation. Mg 2+ -activate the enzyme at a low concentration (0.01–0.04 M), whereas they inhibit at a higher concentration. The optimum of the activity was found at about pH 8, with a K m value at 43 °C of 2.5·10 −5 M for NADP and 2.0·10 −5 M for 6-phosphogluconic acid. The enzyme is stable at 60 °C, whereas it is inactivated at higher temperatures, with a denaturation half-time of 80 min at 70 °C and 3 min at 80 °C. The enzyme shows a broken Arrhennius plot, with two straight lines meeting at about 50 °C.

Selective and quantitative photochemical conversion of the tryptophyl residues to kynurenine in lysozyme

The synthesis is described of the N‐benzyloxycarbonyldecapeptide tert‐butoxycarbonylhydrazide, which corresponds to the sequence 57–66 of bakers yeast iso‐1‐cytochrome c. The peptide derivative was synthesized coupling two smaller subunits via the Rudinger modified azide procedure.

Relation between structure and function in some partially synthetic ribonucleases S′. I. Kinetic determinations

Abstract Dye-sensitized photooxidation is increasingly used as a tool for the elucidation of the structure-activity relationship in proteins. However, the similarity in the relative rates of oxidation of the susceptible amino acids is a serious limitation on the method. In a recent paper (Benassi et al., 1967), we showed that, in formic acid solution, only tryptophan and methionine are photoreactive, if proflavine is used as the sensitizer: the former is converted to kynurenine, the latter to methionine sulphoxide. Since methionine sulphoxide is easily reverted to methionine by reaction with mercaptans (Hofmann et al., 1966; Jori et al., 1968), the possibility exists to perform selective photooxidation of the tryptophyl residues. The method was successfully applied to model peptides (Benassi et al., 1967), however, its feasibility in the case of proteins could only be evaluated through actual application. This report details our findings about the photodynamic action of proflavine on lysozyme (N-acetylmuramide glycanohydrolase, EC 3.2.1.17). Lysozyme was chosen since its threedimensional configuration has been elucidated (Blake et al., 1967), and the chemistry of the enzyme has been thoroughly investigated.

Paramagnetic metal ions as protectors of selected regions of protein molecules from photodynamic action.

Abstract For a better understanding of the structure—function relationships in the ribonuclease molecule, detailed kinetic studies have been carried out on partially synthetic ribonucleases in which some amino acid residues were substituted or deleted. The following ribonuclease S′ analogs have been examined: [Orn10]-ribonuclease S′; des-Lys1-[Orn10]-ribonuclease S′; des-Lys1, Glu2-[Orn10]-ribonuclease S′; des-Lys1, Glu2, Thr3-[Orn10]-ribonuclease S′ and [Pro6, Orn10]-ribonuclease S′. In order to regenerate the arginyl residue, which is present in position 10 in the natural sequence, the S-peptide analogs belonging to the [Orn10]-series were transformed into the corresponding guanidinated derivatives by treatment with O-methylisourea. The activation curves of the S-protein with varying amounts of the S-peptide analog, before and after guanidination, against different substrates have been determined. Moreover, the kinetic parameters of the modified enzymes obtained by mixing S-protein with different S-peptide analogs were calculated. The hypothesis of the existance of an interaction between the γ-carboxyl group of glutamic acid in position 2 in the S-peptide sequence and the guanidinium group of arginine in position 10 has found experimental support. The absence of this interaction, which probably stabilizes the conformation of the N-terminal sequence of the enzyme, brings about some conformational changes on the active center region.

Solution properties of synthetic polypeptides. light scattering and viscosity of poly-γ-ethyl-l-glutamate in dichloroacetic acid and trifluoroethanol

Abstract The paramagnetic ion Co2+ and the diamagnetic ion Zn2+ were coordinated with the glutamyl-35 and aspartyl-52 residue at the active site of lysozyme. Upon irradiation with visible light in the presence of proflavin, Zn2+-lysozyme underwent total loss of enzymic activity as well as of the tryptophyl and methionyl side chains; the kinetics of photoinactivation and of tryptophan photooxidation were coincident with those determined for native lysozyme. On the contrary, irradiation of Co2+ lysozyme caused the specific modification of the peripheral tryptophan-63 and tryptophan-123, and a decrease of the enzymic activity to 84%. The photooxidized sample showed no appreciable distortion in the spatial conformation with respect to the unirradiated protein, thus explaining the retainment of a high catalytic efficiency. The strong inhibitory power of paramagnetic ions against the photodynamic action of dyes on proteins was also evidenced by our studies with ribonuclease A, whose complexes with Zn2+ or Cu2+ were subjected to proflavin-sensitized photooxidation over the pH range 5.8–8.2. At all pH values Zn2+-ribonuclease A displayed a marked photolability, whereas the presence of Cu2+ minimized or completely prevented both the impairment of the biological activity and the damage of the amino acid side chains. In particular, after irradiation at pH 6.9, we isolated a 72% active sample which was modified at methionine-79 and histidine-105. Conformational studies on the photooxidized derivatives of lysozyme and ribonuclease A, and the analysis of our results on the basis of the X-ray models of the native proteins demonstrated that the inhibitory action of Co2+ and Cu2+ occurs throughout almost the entire protein molecule, the efficiency of the protection being related to the distance of a given amino acid from the paramagnetic ion and the symmetry of the magnetic field. Besides providing a valuable tool for protecting enzymes from photodynamic inactivation, the aforesaid technique opens new prospects in the field of dye-sensitized photooxidation of proteins.