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zuo zuo suan tuo qing mei de yan jiu . mei yu fu ji xing zhi de jin yi bu guan cha
Pages: 174-184
Year: Issue:  2
Journal: Acta Physiologica Sinica

Keyword:  琥珀酸脱氢酶酶活力胰凝乳蛋白酶乙二胺四乙酸胰蛋白酶吸收光谱辅基异咯嗪透析水溶性;
Abstract: Purified succinic dehydrogenase is a metallo-flavin-adenine protein containing non-haematin iron.The flavin-adenine prosthetic group is firmly bound to the protein part of the enzyme and cannot be split from the latter by boiling in weak acid medium.By digesting with trypsin and chymotrypsin,however,the prosthetic group can be liberated in combination with a peptide chain.The product has been purified by a procedure which involves cresol extraction, mercuric sulphate precipitation,decomposition of the latter with hydrogen sulphide,followed by paper electrophoresis and paper chromatography.The purified product has been separated into four flavin-adenine peptides with different amino acid contents.One fraction with comparatively high mobility on paper electrophoresis and containing 12 amino acids(hydrolyzed in 6 N HCl) has an absorption spectrum with maxima at 265,350 and 450 mμ(compared with 260,375 and 450 mμ of FAD),the ratio of E_(260) and E_(450) mμ is equal to 3.87.The other three fractions has similar absorption spectra as that of the first,except for a slight shift of the 265 mμ maximum to 270 mμ.All the four flavin-adenine peptides contain cysteine and show a greenish yellow fluorescence in the u.v.light.The fluorescent intensity of the prosthetic group varies with pH and exhibits a maximum at pH 2.9.All fractions are inactive in the D-amino acid oxidase test and give on analysis 1 mole of adenine and 2.5 moles of phosphorus per mole of flavin.The pentose flavin ratio was much higher than that of FAD. Photolysis of the flavin-adenine peptides in alkaline solution yields a product which is insoluble in chloroform after acidification.Removal of the adenine results in the formation of flavin peptides.These facts indicate that the peptide chain is linked to the isoalloxazine nucleus of the prosthetic group.It is known that the absorption peak at 375 mμ of either FAD or FMN shifts to about 355 mμ at pH 12 due probably to the enolization of the keto group at the 2 or 4 position resulting in a redistribution of double bonds in the isoalloxazine ring system. In contrast our flavin-adenine peptide has the corresponding absorption maximum at 350 mμ which shows little positional shift at pH 12.This seems to suggest that the linking of the peptide chain to the isoalloxazine nucleus affects the enolization of the-NH-CO-,which may probably be the site of the linkage. The iron content increases with the specific activity of the enzyme during purification.Iron in the purified enzyme is present in the reduced state.It is firmly bound to the enzyme and can not be removed by prolonged dialysis against phosphate buffer or tris-hydroxymethyl-amino-methane buffer.Enzymatic activity is lost during prolonged incubation with o-phenanthroline or α,α'- dipyridyl and can not be recovered by incubation with Fe~(2+) or Fe~(3+).These experiments de- monstrate a close relationship between enzyme activity and the iron present in the enzyme molecule. The enzyme activity is lower in borate than in phosphate buffer.When 40 mM ethylenedi- aminetetraacetic acid is added to the borate buffer,the enzyme activity is raised almost to the level of that observed in the same concentration of phosphate buffer.The effect of EDTA and phosphate,when present together,is somewhat higher than of either alone.Alanine has a similar effect'as EDTA.
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