THE OXIDAHON OF REDUCED NICOTINAMIDE ADENiNE NNUCLEOTlDE BY ENZYMES FRGM WWII“: mm 311033: fies the Mm a? M. D. MECHIGAN 31’4“? UNIVERSE‘E‘Y films: A. Walks: 19363 {THESlS (:1 —-—-—-—-'. In... LIBP‘DV MlChlgd; . ltc Universi I. / MICHIGAN STATE UNIVERSITY EAST LANSING, MICHIGAN ABSTRACT THE OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE BY ENZYMES FROM LACTOBACILLLLS CASEI by Glenn A. Walker Crude sonicates of Lactobacillus casei have been shown to exhibit NADH oxidase. peroxidase and diaphorase activity. By means of ammonium sulfate fractionation. Sephadex filtration. and DEAE—cellulose chromatography. we have been able to purify the diaphorase some ninety-fold. At this purity. it is completely free of NADH oxidase and NADH peroxidase activity. The properties of this purified enzyme have been studied. The major findings may be summarized as follows: FMN is necessary for maximal activity; benzoquinone, 2.6- dichlorophenolindophenol, and ferricyanide act as electron acceptors while methylene blue does not: the enzymatic rate is not appreciably affected by N—ethylmaleimide. cyanide. amytal or EDTA. Attempts to separate the oxidase and peroxidase from one another by a variety of methods were without success. However, it was possible to study each of these enzymes Glenn A. Walker separately in the semi—purified state. The NALJ oxidase was found to require FAD as a co- factor: the enzyme declined in activity during isolation but could be reactivated by incubation with FAD and cysteine. In addition to oxygen. substrate-level concentrations of FAD or methylene blue could be utilized as electron acceptors. Using methylene blue as acceptor. the enzymatic rate was not affected significantly by p-hydroxymercuribenzoate, N-ethylmal- eimide, cyanide. amytal, or EDTA. The NADH peroxidase was found to bind its flavin component very tightly. Conditions sufficient to remove the flavin resulted in denaturation of the enzyme activity. The native enzyme was unaffected by added FAD or FMN. and was not found to catalyze reaction with any electron acceptor other than peroxide. It is inhibited rather strongly by p—hydroxymercuribenzoate and by N—ethylmaleimide. but not at all by the other inhibitors tested. As was the case with the other two enzymes. the peroxidase was found to be specific for NADH. A maximum activity of seven percent of the NADH rate was found with NADP H . TL.) UXLJNTIQI} OF .LiihKILD IIICQZ’IZ"JLIDE IDLITIKE wwmclwu BY b...43'1‘¢...5 RC)” Lfiamf “an” Pm ‘pp, ‘U~'MOJI Jedi-pd 4-\ 4. fi ‘ I} by Glenn A. Walker A TEESIS Submitted to Michigan State University in partial fulfillment of the requirenlenta for the degree of DOCTOR 0P PHDJOESOE’H" Bepartment of Chemistry 1963 To Lois ACIxTNOWLEDGPENTS The author wishes to express his thanks and deep appreciation to Dr. Gordon L. Kilgour for his guidance‘ and assistance in making this thesis possible. The author also wishes to express his gratitude to Mrs. Charles McCallum for her technical assistance. Finally. gratitude is due the National Institutes of Health for financial assistance during this project. ii INTRODUCTION 0 o o o o o . . HISTGRICRL . . . . . . o o . Diaphorase Peroxidase Oxidase s3mnszressrm . . . - - - ' ’ Enzyme Assay Procedures Materials Chemical Determinations Cell Growth Isolation Procedures Cell Rupture Nucleic Acid Removal hmmonium Sulfate Fractionations Sephadex Columns DEAE—Cellulose Columns Calcium Phosphate Gel Separations Heat Denaturation A Typical Purification Procedure REfiULTE RED DISCUSSION 0 o 0 Isolation and Purification of the Enzymes Diaphorase Peroxidase and Cxidase Properties of the Diaphorase Demonstration of N383 Oxidation Cofactors Acceptors Inhibitors iii .~§9¢-~»‘ TABLE OF CORT .;.~a.\ l .3 22 23 24 25 29 32 34 38 33 38 44 45 45 47 49 51 Page Molecular Weight 52 Stability 53 Properties of the Peroxidase 53 Demonstration of sass Oxidation 54 Stiochiometry of the Reaction 54 Spectrum of Enzyme 56 Cofactors 58 captors 58 Inhibitors 60 Stability 60 Properties of the Qxidase 61 Reconstitution of Activity 61 Acceptors 62 Inhibitors 62 Stability 63 S UMHARY O O O O O O O O O O O O O O O O I O O O O I O 6 4 LITE RATURE CITED 0 I O I O O O O O O O O O O O O O O 0 67 iv Tab ‘- Table 2. 3. 4. 5. 6. 8. 9. LIST OF TRBLES Calculation of ammonium sulfate aaturation............. Purification of diaphorase . . . . . . . Diaphorase inhibition studies . . . . . . Heat stability of diaphorase . . . . . . Stoichiometry for the peroxidase reaction Electron acceptors for peroxidase . . . . Heat stability of the peroxidase . . . . Reconstitution studies . . . . . . . . . Heat stability of the peroxidase . . . . 44 52 S3 56 59 60 61 63 10. ll. 12. LIST OF FIGURES Summary of definitions . . . . . . . . . . . . Gradient elution apparatus . . . . . . . . . . Schematic diagram of enzyme isolation procedures . . . . . . . . . . . . . . . . Typical separation obtained on Sephadex G—100 columns under optimal conditions . . . . . Typical separation obtained on Sephadex G—200 COlumns O O I O I O O O O O O O O O O I I Typical separation obtained on Sephadex G-lOO columns under sub-optimal conditions . . . Typical separation obtained on DEAR-cellulose columns . . . . . . . . . . . . . . . . . Illustration of the stoichiometry of the diaphorase reaction . . . . . . . . . . . Relationship between enzyme concentration and rate for the diaphorase . . . . . . . . . Reciprocal plots for determination of Michaelis constant for FMN’with purified diaphorase . . . . . . . . . . . . . . . . Illustration of the stoichiometry of the peroxidase reaction . . . . . . . . . . . Spectrum of the peroxidase . . . . . . . . . . 31 37 39 40 42 43 48 50 55 INTRODUCTION Variability in strength of binding of flavin co- enzymes to various enzyme protein: is well established. The relative ease of dissociation of D—amino acid oxidase (l) or of NhDPH-cytochrome o roductase (2) contrasts with the covalent bonding of flavin and protein as found in dihydroorotic dehydrogenase (3) and in succinic dehydro- genaoe (4). waever. aside from the reports of Hnennekens and Kilgour (5) and of DeLuca and Kaplan (6) that several analogs of anal wOuld not replace FAD as cofactors for _hog kidney D—amino acid oxidase. there has been little done in any systematic'way to study the different‘binding characteristics among various flavoproteins and the consequence of these differences. In 1950 Snell and Strong (7) introduced a standard microbiological assay for riboflavin using the organism Lactobillu§_g§§§i, It was shown that 2. page; was specific ‘for riboflavin. "‘ ‘ 1The following abbreviations are used: HED‘ and “ADE for oxidized and reduced nicotinamide adenine dinucleotide: NADP+ and NADPH for oxidized and reduced nicotinamido adenine dinucleetide phosphate; indophenol for 2.6-dichlorophonolindo. Phenol: FAD for flavin.adenine dinucleotide: PEN for flavin rel re fu 1&5 “WE-.9... .1“.- .m— IC In 1953. Snell g; gl. (8) found that the closely- related organism Lactobacillue lactig was capable of growing on lyxoflavin without converting it to riboflavin. Heunnekens mg ml. (9) then demonstrated that g, lacgis actually incorporated the lyxoflavin into the cor- responding mono— and dinucleotides. designated LMH and LAD respectively. They also showed that these nucleotides functioned as flavin coenzymes in the cell and could act as coenzymes for NADPH cytochrome 0 reductase and D—amino acid oxidase. although with reduced efficiency. These differences in ability to utilize lyxoflavin for growth suggest either that the flavin-nucleotids-synthe- sizing enzymes of L, Egggi are incapable of acting on lyxoflavin. or that the flavin enzymes of this organism differ markedly from those of g. lactis. Preliminary eXperiments have shown that the majority of the flavin content of an L. gaggi,sonicate is precipitated out in the 40-80% ammonium sulfate fraction. whereas a much smaller percent of the flavins of g. lactis w are precipitated with the protein under the same conditions. mono-nucleotide: LAD for lyxoflavin adenine dinucleotide: LMN for lyxoflavin mononucleotide: DEAB cellulose for diethylamino—ethyl-celluloset and EDTA for ethylenediamine— tetraacetate. the puri Herc 3 the rest appearing in the supernatant solution. In View or these differences. the isolation and purification of the major flavoproteins of both organisms were undertaken. ' This repcxt will describe the work on the £9 3% enzyme-o Strittmatter (10) has shown that a major portion of the flavin—linked oxidativa activity of 12;. we; is due to enzymes oxidizing reduced pyridine nucleotides. Before beginning the historical Section. there are several problems in the area of reduced-pyridina-nucleotide- oxidizing enzymes which should be discussed at the beginning of this thesis. There has been much confusion concerning the terminology of flavoprotaina which catalyse the oxidation of reduced pyridine nucleotides. The terms RADPB or men oxidase. diaphorasa. BABE! dehydrogenase. and mix-cytochrome c reduotasa do not seen to have an exact meaning. As a result they are otten used by different workers to denote quite dittarant enzymatic activities. ‘ In order to avoid most of this ambiguity. the following terminology will be used in this paper: (The basic definitions are those at Dolin (11).) l. P HAD 0:: a Flavin enzymes that oxidize reduced pyridine nucleotides using molecular oxygen 2. 3. 4. as the hydrogen acceptor and producing water or hydrogen peroxide as by—products. Diaphorasex Originally this term designated the flavoprotein which Straub isolated in 1939. but today it has come more generally to mean any tlavin ensyme which catalyses the oxidation 0! reduced pyridine nucleotides by artificial oxi- dants. such as ferricyanide. methylene blue. etc. In many cases the physiological hydrogen acceptors. it any. are not‘known. Huennekens ggugl. (12) have made one distinction in regard to the tenn«diaphorase. They pointed out that it the dye is autooxidizable. as in the case of nethy- lane blue. then the diaphorase can be classified as an oxidase. with the dye serving as an electron carrier. gyggghrome g ggductgsg: Flavoproteins which.cata- lyse the oxidation at reduced pyridine dinucleotide using cytochrome c as acceptor. Many of these enzymes show some 'diaphorsse" activity to arti- ficial oxidants. ‘Qghxfiggggggéggt These are flavoproteins that couple the oxidation of reduced pyridine nucleo- tides to physiological substrates other than 5. 6. cytochrome. or molecular oxygen. 1.0.. non-terminal oxidation. A tyyical physiological substrate might be Coenryme Q. irec . . .' ,u5dases: These are flavoprotein- enzyme: that couPle the oxidation of ”non-coenzyme' substrate: to the reduction of molecular oxygen without involving pyridine nucleotides an inter- mediates. Flavogr_ i P roxidase: These are flavoproteinn which couple the oxidation of reduced pyridine nucleotide to hydrogen peroxide reduction. The above enzymes are illustrated in riguro 1. Another problem encountered in thin area is what‘will be referred to a: 'multipl. activities.“ As was mentioned under cytochrome c reductaees. these enzymes exhibit diaphoraee activity toward some artificial oxidants. These are not the only enzyme: which exhibit this activity. Other tiavoproteins which exhibit diaphorase activity in addition to the classic diaphorase are: NADR oxidaae £rom.beet heart (12): NADPfircytochrome 0 reductase from pig liver (13): xanthine oxidaae from milk (14): and NADHhcytochrome b reductase from liver microcomos (is). This duality of activities has presented several acoaoacauon Mo hnuaEsm a onmmwm noaunamnon Hooamoaoamhnm.homacomOncwson cowhxo \ 38:6 coaxonoo \\\n|\\\\\\\\\\mouunpmnsm nonpo nomoghm nllloluiocfinonoh 523m / / OUdBOOHOSQ ochthm coodcom . ononocmaan o eaonnoouho .llldudflzcom / . . nohn interesting questions.. First. is there actually one enzyme with two activities. or are there two closely related enzymes? Second. if there is only one enzyme with two activitiee. then is one activity real. the true physiological one. and the other an artifact. a product of the isolation procedure? For example. are the cytochrome c reductases one enzyme with two activities--cytochrome c reductase and diaohorase~-or are they composed of two separate but relatively inseparable enzymes? There has been much speculation concerning the diaphoraeea. since the physiological hydrogen acceptor. if any. is not known. Mahler (16) has suggested that diaphoraee is a degraded form of cytochrome reductase. 0n the other hand. Massey (17) has found that Streub's diaphorase ie a potent lipoyl dehydrogenaee. Thin was demonstrated by a constant ratio or the two activities during purification. byinhibition studies and by demonstration of the rapid reoxidization of reduced diaphorase by lipoic acid. There- fore. he suggests that this could be the true physiological role of diaphoraee. In 1939. Straub (18) isolated from pig heart muscle a soluble flavoprotein which coupled the oxidation of menu to methylene blue or indophenol. The enzyme was named dia— phoraae: it showed no cytochrome c reductaee activity. The (lavin prosthetic group was shown to be FAD. 8 In l952 Mahler. gt 51;. (19) isolated from pig heart mmecle a eoluble flavoprotein which ocupled the oxidation of HRDH to cytochrome c reduction. The enzyme. as stated earlier. exhibited diaphoraae activity. The flavin pros~ , thetic group was identified as a flavin dinucleotide not -identical with FAD. In 1957 deBernard (20). using the same method of extraction as Mahler. obtained a man dehydrogenaae front the electron transport particle of heart mitochrondria. The solubilized flavoprotein catalyzed the oxidation of HADHTby'both cytochrome c and ferricyanide. Again. the flavin portion of this flavoprotein was reported to differ‘ from PAD. In 1959 Green at al. (21) isolated. from beef heart mitochrondria. a lipoprotein having Nhnflidehydrogenaee activity using ferricyanide an oxidant. The prosthetic group of this enzyme was FAD. The enzyme was found to be essentially inactive with cytochrome c as an electron acceptor. Green's group found that under appropriate conditions the lipoflaVOprotein could be converted to a flavoprotein with properties indistinguishable from those of Straub‘e . flavoprotein. The conVeraion involved the loss of the bound lipid. They were also able to show that under different conditions their lipoflavoprotein could be converted to the deBernard or Mahler enzyme. This conversion involved not only loss of lipid but also chemical modification of the prosthetic group. Therefore. it seems that the properties of some of these solubilized flavoproteins depend upon isolation procedures. The answers to such probleme. particularly with regard to mitochondrial enzymes. are still some distance away. It is as well. however. to bear some of the possibilities in mind when discussing any flavoprotein electron-transfer system. HISTORICAL To the present time there have been very few reports concerning purified NAnaioxidiaing enzymes as such. In 1955. Dolin reported (22) that 5 reptggoccgg.g§§g§;ig contained several oxidizing enzymes for MADE. These enzymes utilized oxygen. cytochrome c. peroxide and indophenol or fericyanide as oxidants. In 1957.,Dolin reported (23) the isolation of one of these enzymes. the peroxidase. In 1950. Dolin and wood reported (24) the purification of the diaphorase. Both of these enzymes were tlavoproteino. the first requiring FAD as the prosthetic group; the second. FMN. In 1959 Strittmetter (25). using a crude aonicate of Lactobagillua case'. reported the presence of a NADH oxidaae. NfiDH peroxidase. and HAD?! diaphorase. In 1959 Lightbown and Kogut (26. 27) reported the presence of Rich oxidaae. NADH peroxidase. and NADH dia- phorase activity in iysod cells of Bacillus gubtilis. They were able to isolate and purify the diaphoraee; the oxidaee and peroxidase could not be separated from one another. Since we are dealing with three enzymatic activities. the remainder of this historical review will be divided into 10 11 three corresponding part8. Qiap. homes: The first diaphornee was isolated and purified by Straub (18) as mentioned previously. In 1951 Robinson and Mills (28) reported the separation from s eonicato of Paeteurellg gulareneig of four dinphoraces. A NAD3-apeciric diaphorase was obtained from the particulate fraction. It was stimulated by menadione. From the super- natant or soluble fraction three other diaphorases were obtained: 1. A NADPH—specific diaphorase 2. A NADPa—spceitio diaphoraoe ‘3. A non-specific diaphorese In 1958. Stein and Kaplan (29) studied the distri- bution1 of diaphoreses in rat—liver cytoplasm. They found that both the particulate and coluble fractions contained high levels of RADPHB nnd unnfiwdiephoraee activity. similar results were reported by Ereter (30). In 1961 Giuditte and Strecker (31) isolated and purified s water-extractable diaphoreee from or brain. 1There have been numerous reports in the literature concerning histochemical studies on the distribution of diaphorese. These are beyond the scape of this review. 12 This enzyme catalyzed the oxidation of NADPH and NADH'by methylene blue. ferricyanide. 2.6-dicholorphonolindophenol. menadiono and vitamin K . It would not react with cytochromea. l iipoic acid or coenzymo 010. The prosthetic group was FAD. Peroxidase Most of the peroxidase: which have been isolated belong to the class of heme-proteins. They are conjugated proteins which have an iron-protoporphyrin structure an a prosthetic group. usually with one iron~protoporphyrin per molecule. This type of peroxidase is present in most plant tissues: for example. Theorell (32) isolated and crystallised a peroxidase from horse radish. while absoya (33. 34. 35. 36) has carried out extensive etudica on a peroxidase from turnips. Peroxidases have been reported (37) to be able to use some fifty-four compounds as electron donors. The latter can generally be classified as aromatic amines. phenol compounds. aromatic acids and other miscellaneous nubstances such In ascorbic acid. NADDI. ferrocytochrall'c. NADH. etc. The NADH'peroxidase present in g, gg§§;_does not seem to belong to this general class. In 1959. Strittmntter (38). working with a crude preparation. found no heme l3 component. The prosthetic group for the peroxidase wee e flavin compound. which was not further identified. The NADH peroxidaaea studied by Lightbown. £5,5L, (26) and Dolin (23) were shown to have FAD an e prothetic group. Neither group reported the preeenoe or any hem. component in their preparations. These appear to be the only examples of non-heme flavin-requiring peroxidaeee re- ported in the literature. gridase The first reduced pyridine nucleotide oxidese wan isolated from yeast in 1932 by Warburg and Christian (39). They called the enzyme simply "yellow enzyme.” The pros- thetic group of the ”yellow enzyme" was determined by 1 Theorell (40) to be FMN. It is of historical significance that this ’yellow enzyme” was the first flavoprotein to be isolated. Varburg's oxidaee catalyzed the oxidation of HADPH by either molecular orygcn or met ylene blue. In 1933 Haas (41) found in yeast a specific FAD— requiring protein that catalyzed the oxidation of RADPE‘by w—v lIt ehould be noted here that Warburg and Christian demonstrated that the apoonzyme of their enzyme could be combined with PAD to form a flavoprotein which catalyzed the oxidation of RRDPH by molecular oxygen. 14 ' molecular oxygen.. This protein was named ”new yellow enzyme“ in order to distinguish it from the ENE-containing enzyme of Warburg. In 1952. Conn g§.§;. (42) reported that wheat germ contained an enzyme system. NADPH oxidase. which catalyzed the oxidation of NRDPH'by molecular oxygen. They found that the system contained at least two proteins. one or which was a peroxidase. There have been several reports (43. 44) that H333 and NADPH.could be oxidized by\peroxidases in the presence of manganous ions and oxygen. Several flavoproteins have been reported in the literature which possess HRDH oxidese activity as a ”secondary” actiyity. For example. in 1930's xenthine oxidaae was isolated tron milk (45). Thin metailoflavoprotein catalyzed the oxidation ofi hypoxathine to xanthine. It has been, found (46) that highly purified preparations of this enzyme catalyze the oxidation of DPNH. Alec. Friedmann and Vennealand (3) found that their crystalline dihydroortic dehydrogenase possessed NADH oxidase activity. In 1955. Huennekona.g§‘gl. (12) purified 3 Nina oxidaeel from pig heart. This enzyme catalyzed the oxidation ' 1This enzyme could also be classified as s diaphorase on the basis of the definitions given in the Introduction. 15 of DPNH by molecular oxygen in the presence of small amount- of methylene blue. A naturally occurring cofactor was demonstrated but not identified. Reports of the presence of NADH oxidasea in 352. W, g. gubtilis and 1.9,. $9.19.! have been discussed earlier. so dctails will not be given here. In 1962 Mackler g; 3;. (47) reported a fifty-fold purification of the NADH oxidaso from 33.. gggcalig. The enzyme was found to be a rim—requiring enzyme. In 1961 rujui (48) reported crystallization of a HADH oxides. from 20 W. No dotails are available at present concerning this crystallino oxidase. .N‘I"'.‘ ‘ '1 o-'\.. 1“ r...:.‘.-,--..I..£:o.."i. l. 1'3 mam "‘3 s 33 :3; 2'3 3:0 cadence: -.. . - n J”. a. All annoys were carried out either in 1.5 milliliters silica cello or standard three milliliter eilica cells fused into a standard Thunberg tube (both supplied by Pyrocell manufacturing Corporation). Both cells had a: light path of 1.0 centimeters. Changea in optical aboorbenoe were measured by using a Beckman BU spectrophotometer equipped with a log converter (Ledlend Instrumental Engineering} and recorder. Toe standard assay for NADH oxideae consisted of the following: 40 micromolee of phosphate buffer. pH 7.0: 0.13 micromolee of HADH: 0.05 micromolee of PAD: and enzyme. in a final volume of one milliliter. The reactions were started by the addition of the enzyme. 0 The standard assay for diaphoreee activity consisted of the following: 40 micromoles of phosphate buffer. pH 7.0: 0.13 micromolee of race; 0.05 micromolea of FEE; 0.5 micro~ moles ferricyanide: and enzyme. in a total volume of one milliliter. The reaction was started either by addition of enzyme or ferricyanide. 16 17 The standard assay for peroxidase activity consisted of the following: 40 micromole: of acetate buffer. pH 5.4: 0.13 micromoles of HADH; 0.9 micromoles of hydrogen peroxide; and enzyme. in a final volume of one milliliter. The reaction was started by addition of enzyme. The oxidation of HKDR in each case was followed by the measurement t the decrease in absorbancy at 340 mu. For all calculations. a value of 6.22 x 106 cog/hole was used for the molar extinction coefficient for anon (49). One gal; of enzyme activity was defined an that which brings about a change in absorbance at 340 mu of0.010 per minute. Specific activity was then defined as the number of activity units present per milligram of protein. Since we are dcaling with three NADA oxidizing activities it was necessary to assay the diaphorasa and ' peroxidase activity anaerobically during the early stages of purification. The anaerobic assays were the same as the standard. except that three times the amount of each component was unadin a final volume of three milliliters. All of the components except EADH'wero mixed in the main compartment of the cell. The NADH was placed in the side arm.‘ The reaction was started by tipping the Nina into the main compartment. Fraction: obtained during various column separations 18 were routinely nsaayod aerobically for peroxidase activity at pH 5.4 and for diaphoraso activity at pH 7.0. Oxidase determinations were then made at both pH values and the peroxidase and diaphoraso values corrected for oxidation due to oxidaao activity. Oxides. activity of cell extracts was found to decreaae slowly with time during the processes of isolation of the enzymes. A conzidcrable portion of the original activity could be restored by incubation of the inactive enzyme with cyateine and PAD. Enzyme preparations were reactivated by incubating 0.05 to 0.2 milliliters of enzyme for ten minatos at 37° with 0.1 milliliters of FAD (l x 10.3m) and 0.1 milliliters of cysteine (0.242 grams of free base dissolved in 50 milliliters of 0.5 M.potaasium phosphate buffer at pH 7.5). The assay for the reconstituted oxidase consisted of the following: 40 micromolen of phosphata buffer pH 7.0: 0.13 micromolea of NfiDH; and reconstituted enzyme in a final volume of onn milliliter. materials- The following materials were used: FAD. Ffifl'cyto- chrome c and NADH. Sigma Chemical Comnany: catalaae. alcohol dehydrogenase. ribonucleaaa and deoxyribonuclease. Worthington: l9 Sephadex 6—25. 6-75. 6-100 and 6-200. Pharmacia Fine Chemicals Inc.: methylene blue. Rational Aniline Division. Allied Chemicals; acriflavin. Mann Research Laboratory: amytal sodium. Eli Lilly: manadionc. National Biochemical Corporation: 2.6-6ichlorophcnolindophcnol. Eastman Organic Chemicals: riboflavin. General Biochemicals. Inc.: DEAE- cellulose. Eastman Chemical Company: Nkethylmaleimidc. Sigma Chemical Company: p-hydroxymcrcuribenzoatc. sodium. California Corporation for Biochemical Research. The potassium ferricyanide was recrystallized from boiling water.. The solutions were stored in brown bottles. The p4bcnzoquinone and l.4~naphthoquinonc were recrystallized from light petroleum ether. The p-bcnzoquinonc was dissolved in water. divided into three-milliliter portions. and trozcn. Ho solution was used utter uitting six hours. The 1.4-naphthoquinonc and monadionc were dissolved in one millilitcr or ethanol and rapidly diluted to the desired volunc with water. The p-hydroxymercuribenzoatc was dissolved in the minimum amount of 0.1 M Knox! and then diluted up to the desired volume. mum and BADPH were dissolved in 0.002 M potassium phosphate buffer at pH 7.5. 20 Chemgca; Determinations Protein concentration was determined by the method 1. (50) and/or by 280-260 mu ratios according Into-I. of Lowry g; to Warburg and Christian (51). For quantitative estimation of the following compounds by optical absorbence. the values shown were used for the molar extinction coefficients: FAD. 11.2 x 106 cmz/mole at 450 mu (52): FMN and riboflavin. 12.2 x106 cmz/mole at 450 mu (52): cytochrome c. oridized. 0.96 x 107 7 cmz/mole; reduced. 2.81 x 10 cmz/mole at 550 mu (53): 6 NADH. 6.22 x 10 cmz/mole at 340 mu (49); Fe(CN)6. 1 x 106 cmz/mole at 420 mu (54): 2.6—dichlorophenolindophenol. 2.1 x 107 cmz/mole at 600 mu (55). Cell Growth The medium used for the growth of Lactobecillus casei ATCC 7469 contained the following components per liter: Tryptons (Dirco)' 10.0 grams Yeast extract (Difco) 10.0 grams Dextrose 5.0 grams KZHPO4 2.6 grams The pH was approximately 7. and no adjustment was required. The procedure for the growth of this organism was as follows: All of the ingredients for 18 liters of medium with the exception of the dextrose. was placed in a five 21 gallon pyrex carboy and 18 liters of water was added. Enough dextrose for 18 liters of medium was put in a 500 m1 Erlenmeyer flask and made up to 360 milliliters with water. The dextrose was autoclaved separately. since dipotassium hydrogen phosphate is a catalyst for its caramelization. It can also be heated in a more precise manner in a small volume. 'One and one-half liters of the dextrose-free medium in the carboy Was put into a two liter Erlenmeyer flask and thirty milliliters of dextrose solution was added. Thenlso milliliters and 15 milliliters of dextrose-containing medium was put in a 250 milliliter Erlenmeyer flask and a test tube. respectively. All containers were plugged with cotton. surrounded with gauze. then covered with brown paper and tied with string. The carboys were auto- claved for 1 hour gtter they had reigned-12139 The rest of the flasks were autoclaved for 15 minutes at 121°C. In order to build up a vigorous carboy inoculum. the test tube was first inoculated.trom a stab culture and. after the appearance of visible turbidity. its contents were poured aseptically into the 250 milliliter £1a9k. - This serial transfer of the inoculum was repeated through the two liter flask and finally into the carboy. The remaining dextrose solution was added to the carboy along 22 with the inoculum. The turbidity of the culture in the carboy was checked periodically by sight and when no further increase occurred. the contents were harvested in a Sharpie: continuous centrifuge. The growth period in the carboy was about 16-20 hours depending upon the number of organisms in the inoculum. Isolation Procedures gel 1 Rupture Several procedures were attempted for cell rupture. Breakage was attempted first in a model 45 Virtis Homogenizer. It was found that suitable breakage could be obtained only in the presence of glass beads. The major problem encountered with this procedure. however. was‘keeping the Virtie tlask cooled. The flask was packed in ice and the breakage carried out in the cold room. Still. the heat generated in the presence of the glass beads was not conducted away fast enough. so that the solution warmed to forty or fifty degrees Centigrade. he a result the procedure was abandoned in favor of sonic oscillation. Variations in temperature. time. glass bead concen- tration. etc. were tried with the sonic oscillation process. The following procedure appeared to give the beat results: After harvesting. the whole cells were suspended in phosphate 23 buffer pH 7.0. 0.02 H to give a thick slurry. This slurry was subjected to sonic oscillation in a Raytheon 10 kc sonic oscillator for 55 minutes. The sonicate was removed and diluted with the above buffer. This was followed by repeated centrifugation at 28.000 RC? in an International Karl centrifuge until the supernatant solution was almost clear. -The debris was made into a slurry again with phosphate buffer and subjected to a further period of sonic disruption. The supernatant solution was cleared bf repeated centrifu- gation at 28.000 RCF. An example of a_typical run would be as follows: 154.2 grams of wet L. casei (corresponding to 49.3 grams. dry weight) were mixed with forty milliliters of phosphate buffier. The resulting slurry was disrupted by sonic oscillation as outlined above. The final volume of the supernatant solution was 360 milliliters. which contained approximately 18 milligrams of protein per milliliter of solution. prucleic Acid Removal The sonic supernate contained a high nucleic acid content. Using 280 mu to 260 mu ratios according to sarburg and Christian (51). the nucleic acid content was calculated 24 to be in the range of 50 to 60%. The nucleic acid was enzymatically hydrolyzed by incubation with deoxyribonucleaso and ribonuclease. Two to five milligrams each of ribonuclease and deoxyribonuclease were added to the sonic supernatant. the exact amount de— pending upon the volume of the solution. This solution was then incubated in a low-actinio flask at 37°C for six to eight hours. Similar results could be obtained by allowing the flask to sit overnight at room temperature. Upon incubation a precipitate usually formed. which was removed by centrifugation. The resulting supernate was a clear yellow solution. The nucleotides which resulted from hydrolysis were removed during the ammonium sulfate fractionation and Sephadex columns stages of purification. Ammonium Sulfate Fractionation: All ammonium sulfate fractionation procedures were carried out at 0°C. The percent-saturation values were calculated from the data in Table l. The calculated number of grams of finely-ground solid ammonium sulfate were then added to the solution with gentle continuous stirring. After stirring for forty-five minutes. the precipitates were spun down in a refrigerated centrifuge by spinning for 15 minutes at 18.000 ESP. 25 Calculation of ammonium sulfate saturation.l Table 1. Temp. 0° 10° 20° 30° Molarity of oat. soln.3.9 4.0 4.1 4.2 Salt Content in Wt% (G) of (NH 1 so Soln's of Various o 4 2 4 Molaritiea (m) at 20 m 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 G 1.3 2.6 3.8 5.1 6.4 7.6 8.8 10.0 11.1 12.3 m 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 G 13.4 14.6 15.7 16.8 17.9 19.0 20.1 21.2 22.2 23.3 m 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 G 24.3 25.3 26.4 27.4 28.4 29.4 30.3 31.3 32.2 33.2 m 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 G 34.2 35.1 36.1 37.0 38.0 38.9 39.8 40.7 41.6 G (loo-o ) . .2. l .at. Formula: X ‘V [61(100-6 ) 117.6 2 but when 61 - 0 one uses 6 ._.;E._ X ' [100-6 1 v 2 1 G. Beieenherz. 35 $1,. 2. Naturfarsch.§§. 555 (1953). . .4 , H ..r 26 Various fractionations were attempted. The three enzymes under study were found to precipitate in the 40-75% precipitate fraction. Therefore. the best fractionation seems to be 0-40. 40—75. and 75-ioes. In some of the runs 0-50. 50—80 and 80-100% fractionations were used. In these cases parts of each of the enzyme activities fiound were in the 0—50 fraction: the bulk of the activity. however. was found in the 50-80 fraction. Sephadex Columns Preparation of Sephadex Columngz The dry Sephadex was thoroughly suspended in a dilute sodium chloride solution. The gel was then washed repeatedly by decantation with distilled water in order to remove the finer gel particles and most of the sodium chloride. The preper size chromatographic column was filled 'with distilled water and mounted in a vertical position. The gel suspension was added to the column through a funnel 'which had been mounted in the top of the column. When several centimeters of gel had settled. the outlet of the ‘column.was opened. After the gel had completely settled. a piece of circular filter paper was placed on top of the column to prevent disturbance of the gel surface. 27 The packed column was then equilibrated with 0.002 M potassium phosphate buffer. pH 7.33. The buffer contained eight drops of octanoic acid and eight drops of zephiran chloride solution in each four liters to diacourage bacterial and mold growth. The ”void volumes.” the volume of eolution outside of the gel. of the columns were determined by placing a narrow zone of India ink on the top of each of the columns and eluting with buffer. This elution volume ie equal to the void volume. The internal volume was determined by adding a small volume of sodium chloride solution to the top of the column and eluting with the usual buffer. This elution volume is equal to the void volume plus the internal volume. Also. in some cases the internal volume was calculated from the following equation; V1 8 internal volume a a dry weight of gel V a a x Wr used .. l “r a water regain AA 1 . . Average values for water regain. in grams of water per gram of dry gel. are c_ven by the supplier. J 28 Sephadex»3eperetiog Procedureg: In general. the protein solutions were placed on top of the Sephadex column in a narrow zone. The column was then eluted with 0.002 M phosphate buffer. pH 7.33. Unless stated otherwise. the elutions were carried out at room temperature. Constant volume fractions were obtained by using a fraction collector with drop counter. A typical example or a G-lOO run would be as follows: One tube of the 50—80% ammonium sulfate precipitate was dissolved in phosPhate buffer 0.002 M. pH 1.33. to give a final volume of 4.6 milliliters with a protein concentration of 66.6 milligrams of protein per milliliter.* 4.4 milli- liters of thin solution was placed on a Sephadex G~100 column. 4.5 x 48 centimeters. and the column eluted with the same buffer. The best separation was Obtained when the flow rate was approximately 0.5 milliliters per minute. When the flow rate was faster and/or the protein volume larger. poorer separations were obtained. It was found that the results are highly reproducible: a typical pattern is shown in the section on Results no Figure 4. The general procedure for 6—200 columns is the same as for 6—100. A typical run was carried out as follows: Two milliliters of the protein preparation (which contained 29 33.5 milligrams of protein per milliliter). was placed on. a Sephadex 6—200 column 30 x 2.2 centimeters. The components were eluted from the column using 0.002 H.potassium phosphate buffer at pH 7.3. Three-milliliter fractions were collected. The flow rate was adjusted to approximately 0.5 milliliters per minute. The results are shown in Figure 6 of the Results section. DEAR-Cellulose Colgmns .greparation o£¢§olumnez A weighed amount or dry DEAR- cellulose was dispersed in a Waring Blendor for approximately one minute. A slurry was prepared by adding potassium phosphate buffer. 0.05 M. pH 6.8 to the DEAR-cellulose. This slurry was then poured into a suitable column and allowed to settle. The column was then brought to equili- brium by washing with the same buffer. After each use. the columns were regenerated by washing with this same buffer overnight. Some of the small columns were packed with the dry DEAR-cellulose directly by repeated gentle tamping of small amounts until the desired height was obtained. separation Procedures: The protein solution was usually placed on top of a DEAR-cellulose column which had been pro-washed with 0.05 H.phoephate buffer. pH 6.8. Various 30 elution procedures were used in the attempt to obtain maximal separation of the proteins. Again. unless stated other- wise these columns were eluted at room temperature. A gradient elution system. Figure 2. was set up which consisted of three chambers. The first two were mixing chambers while the third was a reservoir. The best elution tried‘wss the following: Chamber I ~—- 0.05 M.phosphats buffer. pH 6.8 Chamber II --- 0.1 M.phosphate buffer. pH 7.0 Chamber III --- 0.2 M phosphate buffer. pH 7.2 Equal volumes of each buffer were used in a given run. A volume of 250 milliliters of each buffer gave satisfactory results with a column 32 x 1.2 centimeters. A typical run would be as follows: 35 milliliters of the combined fractions from a 6—100 column were placed on a DEAE cellulose column. 33 x 1.2 centimeters. which had been preewashed with 0.05 M phosphate buffer pH 6.8. The column was then eluted by gradient elution. (See Figure 2 for elution apparatus) in the following manner: Chamber I --- 250 milliliters 0.05 M.buf£er pH 6.8 Chamber II --- 250 milliliters 0.10 M buffer pH 7.0 Chamber III--- 250 milliliters 0.20 M‘buffer pH 7.2 Five—milliliter fractions were collected. The results are shown in Figure 7 of the Results section. . ,dlldar... I End. 'l .IHPE (Id r} . uEthw 31 f 356.3%: coapaam €32.93 m charm Law -— I'll: ' HHH Aopfiano 5:300 06 -¢l -—-—l® H .3338 HH hon—nanny 32 (r Bully} P: ‘5 "i5 ‘1' sat"? C“... 3x27 filagfx 7'1r.n.3 The calcium phcegnate gel mas prepared according to the aethcxi of lieilin and fiartree (C 3). &everal difficrent procedures were tried. First. a Mt pte were made to se Mctively adsorb one of due enzyrm e to the gel by addition of varying amounts of gel. The general proced cure emoloyed was as follows: The contincd fra: tisns (280 milliliters) from a G~103 column separation were precipitated by adding ll? grams of solid ammonium Bulfete. The precipitate was stirred for one hour. collected by ccntrifugation. then dissolved in water and dialyzod cver~ night. The dialyzod eolut ion was ad_ju ted with sodium cetate buffer to give alpfi of 5.0 in a final volume of 133 milliliters. which contained 1. 7 milligrams of p; otein per milliliter. Seventy milligien of gel were added with stirring. kite: stirring for twenty minutee the gel wee collected by centrifugation and designated "Gel I.” To the supernatant solution. 165 milligr “me of gel was added. the above steps repeated. and this gel designated ”" wl II." This procedure was repeated twice more to give ":0 ell III" and ”Gel IV.“ Each gel was then eluted in succession with 0.l M.pctaeeium phoeghete bufrers at pH 5.&. 6.0. and 6.8. The procedure for elution was as follows: The gel was suspended in 25 milliliters of buffer (pH 5.4) for five 33 minutes. The gel was than collootoi by cohtzifugotisn and tho supernatant assayed. The gel was then eluted with buffer at pH 6.0 and then pH 6.8. This procedure was repeated for gels II. III. and IV. It wag found that tho peroxidase. oxidase and ferricyanido activities were scattered throughout the elution ategs. Secondly. attempts were mafia to oelectively elute the enzymes from the gel by either changing pH or ionic strength. The general procedure employed was as follows: The dialyzed G—lCO fractions were adjusted with 1.0 M potassium phosphate buffer pH 6.75 to give fiflty milliliters of protein solution 0.001 M in phosphate at pH 6.75. To forty-five milliliters of such a solution. 700 milligrams of gel were afided and the gel was collected by centrifu- gation. The resulting suoernatant solution contalnoa no peroxidase. oxidaso or ferricyanide activitiés. The gel was successively eluted with potassium phosphate buffers at pH 6.75 of the following molarities: 0.005 M. 0.01 M. 0.02 M. 0.04 M and 1.0 M. The elution procedure is the came an that stated above. (In anotherrun the gel was eluted with 0.1 M potassium phosphate of varying 98: 6.8. 7.0. 7.2 and 7.5. In this case the protein had been abaorbed ”3?- '1“! I. 34 at pH 6.0.) It was found that the results again were varied and. most important. they could not be successfully reproduced. Attemgta were made to selectively heat denature one or more of the enzymes. The protein.aolution was heated in a water bath at the following temperatures: 1. 40°C for 10 minutes 2. 50°C for 5 minutes 3. 60°C for 5 minutes 4. 70°C for 5 minutes After heating the solution at each of the above temperatures. the preparation was immediately chilled and any coagulated material removed by oentrifugation. an aliquot was removed for assaying. and the remaining solution wau heat treated at the next temperature. A Eypical Purification Procedure After harvesting the ;. gagg; cells. forty milli- liters of 0.02.M potassium phosphate buffer at pH 7.0 were added to 154 grams wet weight of g, ceuei which corresponded to 49.3 grams dry weight. The resulting slurry was subjected to sonic oscillation for fifty-five minutes. The debris was spun down as stated in Procedures. The final volume of the supernatant solution was 365 milliliters. 35 To 360 millilitere of the sonic supernatant 4 'milligrame each of ribonucleeee and deoryribonucleaee were adaed and the solution was left to incubate at room tewpera- ture overnight. approximately 15 hours. The precipitate which weefound after incubating was removed by centrifugation. The final volume was 345 nulliliters. Ninety-nine grams of solid ammonium sulfate was added to 340 milliliters of the incubated supernatant. The' precipitate was collected by centrifugetion after stirring for 45 minutes. The precipitate was stored at 4°C until further used. Eighty-nine grams of solid ammonium sulfate wee added to the O-SO aunernetant with stirring. after stir- ring for 45 minutes. the precipitate was collected by centrifugation at 18.000 REF. Again. the precipitate was stored at 4°C until further used. Ppproximetely one-tenth of the 50»89%.ammonium sulfate precipitate was dissolved in 0.002 M;potaseium phosphate buffer at pH 7.3 to give a final volume of 4.6 milliliters which contained 66.6 milligrams of protein per milliliter. 4.4 milliliters were placed in a narrow band on a Sephadex 6-100 column. 4.5 x 48 centimeters. The column was eluted at room temgerature with the same buffer at a flow rate of 0.5 milliliters per minute. Five milliliter fractions were collected. The peroxidase and oxidase were located primarily in fractions 45 to 60. while the diaphoraee activity was found in the 64 to 72 fractions. These fractions were combined. The final volume of the combined 45~60 fractions was 59 milliliters. Pifty~four milliliters of this solution was placed on a DEnE-cellulose column. 33 x 1.2 centimeters and eluted with the following gradient: 250 milliliters of 0.05 MLphosphate buffer at pH 6.8: 250 milliliters of 0.1 M phosphate buffer at pH 5.0; and 250 milliliters of 0.2 H phosphate buffer at pH 7.2. The gradient set—up is depicted in Figure 2. Pive~milliliter fractions were collected. The peroxidase and oxideee were found in fractions 36 to 50. The final volume of the combined 64 to 72 fractions was 42 milliliters. Thirty-eight milliliters were placed on a DBAEacelluloee column. 33 x 1.2 centimeters and eluted with the above gradient. The ferricyanide activity was found in fractions 40 to 60. This was used as the "purified diaphorese" for property studies. The purification procedure is outlined in Figure 3. 37 Calls 1. Sonication 2. Centrifugation II I Debris Supernatant RNaaa 1. Incubation DNasc 2. Centrifugation fi Supernatant Precipitata (discard) 1. (11332304 (o-soz eat.) 2. Contrifugation II A ’1 Pracipitata supernatant 2. Contrifugation ll 1 Precipitatc supernato (discard) l. Dissolve in buffer 2. Sephadex 0-100 1 "Peroxidase "Diaphcrasa fractions” . fractions" DEAE- ' DEAR- callulose cellulose Paroxidase- Purified. Oxidaaa diaphorasa Figure 3 Schematic Diagram.of Enzyme Isolation Procedures RE SULT 5 11.119 DI 3C US 5 ION Iaolaticnwgnd Purification of the Enz”me‘ The main objective of this phase of the project was the separation of the three Hana—oxidizing activities of g, gaggi. and the purification of each of these enzymes. A major problem encountered in working with the cell» free sonic supernatant was the high nucleic acid content. on a weight basis. the nucleic acid content was 1.5 times that of protein (an determined from 280 mu - 260 mu ratios (51)). 'After incubation with ribonucleaee and deoxyribo— nuclease.£ollowed by ammonium sulfate fractionation and Sephadex filtration. the nucleic acid concentration was reduced to only one percent of that of the protein. The diaphorase or ferricyanide activity has been successfully separated from the REDS peroxidase and NADH oxidase activities. The best separation was obtained with Sephadex G~100 and 6—200 columns. Ueing the conditions listed under Sephadex Column Procedures in the Experimental Section. complete separation was obtained. as shown in Figures 4 and 5. When the conditions were varied. such 38 113 of protein/m1 or fraction 39 5 5ar .——0 Protein F cp_o Peroxidase v——v Diaphoress +-+ Oxidase ..4 ub3 ..2 --l 90 Fraction number Figure 4 Typical Separation Obtained on Sephadex 6-100 Columns under Optimal Conditions A total or 302 mg or protein in 4.4 ml was added to the column; eluted with potassium phosphate buffer, 0.002 M. pH 7.3. Five ml fractions were collected. 9‘0: x (uoraopag JO Im/catun) Abeorbance 40 2.5! 32.5 2.01” “-2.0 H l.5"{ “F105 100‘? drloo 0.5‘- 1~0.5 Fraction number Fifiure 5 Typical Separation Obtained on Sephadex 0-200 Columns The following symbols are used: absorbence at 280 gnu—e; absorbanoe at 450 mp,+-—+; peroxide se, o——o 3 diaphora as, H. 9.0: x rm Jed aqtun emflzua 41 as a larger volume of column feed and/or a faster flow rate. incomplete separations were obtained: a typical result is shown in Figure 6. It was clear from data such as those shown in Figure 7 that the DEAR—cellulose columns also separated the ferricyanide activity from the peroxidase and oxidase. or more importance. however. was the finding that passage through the DEAR-cellulose procedure also resulted in a considerable increase in specific activity. Thus the figures in Table 2 indicate approximately a nine-fold increase in specific activity for this step alone. with the Sephadex column separation. there is also a ferricyanide peak which corresponds to the peroxidase- oxidase peak. Since this persists in preparations from which the other ferricyanide peak has been removed completely. it is probable that this peak represents a "secondary activity" of either the peroxidase or oxidase. and does not represent incomplete separation. The summary for purification of the ferricyanide (diaphorase) activity is found in Table 2. A 94-fold purification has been obtained with approximately 2.¢% recovery of the total activity. Enzyme units per ml x 10"5 uncapaocoo Hganoupsm moons massaoo ooauo Nonannom :0 ©0536 coaaanapm Havana. o opzmam noose: co.“ poehm OOH 00 cm Ob Ow on on". L.md HI! AI¢OO ..o.o Na: lrmwoO ..o.H nl‘ III. 898.330 I .5...” 3333.3." To fl 5.59"." I :. v; 0..” tux Jed utoaoad 9n 43 or x {w Jed satun emizug I- .onou adopmaaxonaoe one! crane no: open: noaaapapoe cahucm .xeoa omeoauonon on» near coeducaoo mpa>ap :oe omooano muamnuoco hp ooosoon coop open moapapaaoe ooeodxonoa one .rIIJn .ooanosodao «oloJmaoHHvon «I .53th «coo: one goofing wfitoaflou och. acasnoo omoasaaooumamn :o cocaeuno coapehoqom Heoaoha b onswam paces: coapoanm ooa £o \bbp Jw &m we mm &W mm d -‘ 4 d o 4 1 1 1 H4. . miw , a: v i H c; 5.0 do 088 48 eouaqaosqv E'! U".':"‘ 44 Table 2. Purification of diaphorase. Fractions Protein cone. Specific Total mg/ml Activity Activity Original 17.5 85 550,000 Incubated 20.0 80 553,000 52:10? git. 68.7 300 93. coo Q. 4 2 4 Combined fraCtiODB 0.458 920 3“}. 00* Sephadex 6.100 E n.~ , DE“E °°llul°89 0.036 8000 1.440 Free. 45 *Only one-tenth of the 50-83% saturation ammonium sulfate precipitate was carried through the column steps. Peroxidase and Oxidase To the present time it has not been possible to separate the peroxidase and oxidaee activities. Ammonium sulfate fractionation. Sephadex G—lOO. heat denaturation. calcium phosphate gel adsorption. and DEAR-cellulose chromatography have proven unsatisfactory for separation. Dolin (22) has reported that the NADH oxidaee of l§p ggecalia is precipitated in the 0-50% ammonium sulfate fraction. Madkler.‘gt.§;. (47) has taken the 0-50% ammonium sulfate precipitate from.§. fieecalig and purified the oxidaae 50-fold. They reported that the purified oxidaee Ir .. lull. 4H... 45 catalyzed the reduction of oxygen to water. which is dif» ferent from the results of Dolin (22). without the obligatory participation of a NADH’peroxidase. Dolin also reported (23) the purification of the NADH peroxidase from g. faecalis. It is of interest to note that the oxidaee was not assayed according to the procedure E of Heckler £5.9l. (47) for reconstitution. Lightbown and Kogut reported (26) that the peroxidase | and oxidaee activities of Q. aubtilie could not be separated u by any of the procedures which they tried. They were able to separate the diaphoraee from the peroxidase and oxidaee by DEhE-celluloee anion exchange chromatography. Properties of the Diaphgraée Demonstration of N’Dfi'Cridation When the reaction mixture contained NADH. buffer and enzyme. the disappearance of NADH. as measured by optical density at 340 mu. wee very slow. Upon the addition of potassium ferricyanide an appreciable rate was observed. (Point I of Figure 8—-the increase in absorbency is due to the ferricyanide.) fter the reaction had proceeded to completion. the pa*was adjusted to pH 8.5 i 0.5 with NaOH. and 0.1 milliliter of 95% ethanol was added (Point II of Figure 8). Upon addition of crystalline alcohol dehydrogenaee. 1.0Qr 0.8 3‘ Point I 3 o 6 n O '9 G O 0 g r: 0.4 « n o D .9 4 Point III 0.2 4. Point II \ J: Time Figure 8 Illustration of the Stoichi- ometry of the Diaphoraee Ree etion. 47 the absorbency returns immediately to a value corresponding to 90% or the decrease (Point III of Figure 8). (Dolin (22) has shown that 90%.of the decrease in abacrbance at 340 mu in the diaphorase assay is due to NADH'oxidation. and the other 10% to ferricyanide reduction.) This demonstrates that the disappearance is due to the oxidization of the NADH to HAD+ and is not due to destruction of the NADH‘or conversion to a modified NADH product (57. 58). The effect of variation in enzyme concentration is iehown in Figure 9. The enzyme appears to be specific for NADH. The rate with NRD?H as the substrate under identical conditions is only 7%.of that with NNDH. Cofactors When FMN is added to a reaction mixture containing NADH. buffer. enzyme and ferricyanide. the decrease in absorbence at 340 mu is approximately tripled. However. there is no appreciable change in rate when FMN is added to a mixture containing only NADH; buffer and enzyme. The nonenzymatic rate is similarly not affected by the presence of FMN. Addition of riboflavin had no effect upon the rate. The action of added FAD was found to vary with the age of the 13 Absorbance per minute 0.50 ' 0.40“ 0.30q 0.20 ‘ 0.10‘ U 48 1 l 1 J I I 0.02 0.04- 0.06 0.08 0.10 Volume of Enzyme Solution (m1) Figure 9 Relationship Between Enzyme Concentra- tion and Reta for the Diaphorese. Protein concentration in the enzyme solution was sixty micrograms per milliliter. 49 solution. A fresh solution. showing no significant amounts of FMR‘on chromatography in 5% H323P04 solution. gave about a 20% stimulation of rate. Older preparations gave up to 50%»stimulation. but were found to contain traces of PMN on chromatographic analysis. This is a particularly trouble— some problem because 0: the relatively low apparent Km for PEN. An attempt was made to calculate a Km for the INN. In order to make this calculation it was assumed that FMN had been completely split from a portion of the enzyme and that the added PMN‘was reactivating this portion of the enzyme. Therefore; the “inherent“ rate was subtracted from the rates found with varying concentrations of added FMN. Using this approach. Figure 10 gives the l/v versus l/[Sl and [SI/v versus [S] plots. Prom these plots the "apparent” Km was determined as 1.8 x 10‘6M. Acceptogg Various electron acceptors were tested. It was found that riboflavin. excess PEN. and FED are inactive. The purified preparation did not act as an when peroxidase nor as an HADH oxidase. even when the preparation was incubated with FED and cysteine. It is of interest that :methylene blue. which is regarded as a typical flavoprotein 50'l €9x:105 gigugg lO ‘ -%~ —4‘ rfi 100 200 300 400 EQJK 107 q» Reciprocal Plots for Determination of Michaelis Constant for FMN with Puri- fied Disphorsse oxidant. is not an ac tcu:pt or. The best acceptors of those tried were puben;.;.inoue and indophenol. it e»..i c incuiiot.rda. the rate with p—Lcnaoguinone was seven times tnat with fer; wicr 11s; E. The rate with indophenol wee three times that wits ferricve “nice even though the inic hsnol cone cntrativn wee oncntarth that of the rerric genius. Doiin (2;) has simila rly f-ound th at benzoquinone andtoluquinone ape the beat oxidants for the diaphorase from_£. 5""j?ia. fi—ver—fi Table 3 SZOJS the effect of sever al inhibitors upon the ferricyanide actip'itgr. For this at ity the Luf fc r. Eflflfi. FHN enzyme and inhibitor were added to the cuvette. mixed. and allowed to incubate for ten minutee at room temperature. The reaction was then started by addition of ferricyanide. When p-hydroxymercuribensoate was used. an anomalously hi 3h non~enzyma tic rate was obtained with ferr icyanide. Because of this anomalous rate. it was impossible to interpret the p—hydroxymercuribenzoate effects on the enzymatic reaction. as indicated from Table 3. no major degre e 02 specific inhibition is obtained. These results are in full agreement with hose of Dolin (24). 52 Table 3. Diaphorase inhibition studies. m - A L J... L M *M- ‘“ v—v L. ‘ *— —._—. h. —‘r— v v— ‘-——~ '— . v— w Inhibitor CO?§:§:::::?“ % Inhibition Amytal 5 x 10-4 1 x 10-3 H~ethyl maleimide 5 x 10"4 11 x 10‘3 16 tom 5 x 10"4 x l0'3 Cyanide s x 10" 6 x 10“3 17 Hyfirogen peroxide 1 x 10-3 11 A M— - : . A A— . .4 . _ .. .._._ Stanfiard Diaphoraae Assay ~ (NRDH cone. - 1.3 x 10’4M) figleogiaz eeiqgg Because of the limited amount of purified enzyme available. no detailed studies of molecular weight could be carried out by physical uethoés. ouch a3 ultraoentri- fugation and/or electrophoeie. However. using Sephadex results. it is possible to make an estimate of the molecular weight. The minimum weight has to be greater than 25.000 since the protein is not held up on euzs. The maximum weight 53 is less than 75.000 since diaphoraae is held up somewhat on 6—75. This would place it in the range of 30.000 to 70,000. It is interesting to note that Dolin has reported (26) a molecular weight of 50,000 for the diaphoraae from S. faocalis. based on bound flavin determinations. a-umm The enzyme is stable at 4°C for at least a month. The efifects of heating the enzyme at different temperatures are shown in Table 4. Table 4. Heat stability of diaphoraee. Treatment ' $.03 Original Activity 40°C for 10 minutes 100% 50°C for 5 minutes 10Q% 60°C for 5 minutes 70% 70°C for 5 minutes 6% Properties of the For xidaee Although it has not proved possible to separate the oxidaee and peroxidase activities. it has been possible to study the properties of each in the partially purified system. The oxidaae is assayed at pH 6.8. where peroxidase activity is negligible: the peroxidase activity at pH 5.4. where the oxidase rate is only 5 per cent of the peroxidase value. anv- Wan 54 In fact. the slight oxidaea activity at pH 5.4 was a problem only in attempts to study alternate electron acceptors for the peroxidase. It did not interfere in the other studies. ‘ggmonetration o§_fim03 Oxidation When the reaction mixture contained NADH. buffer and enzyme. the disappearance of NADH. as measured by optical density at 340 mu. was very slow. Upon the addition of hydrogen peroxide: an appreciable rate was observed. (Point 1 of Figure 11). After the reaction had proceeded to completion. the pH wan adjusted to pH 8.5 1 0.5'with sodium hydroxide. and 0.1 milliliter of 95% ethanol was added (Point 2 of Figure ll). Upon addition of crystalline alcohol dehydrogenaee. the absorbency returns immediately to a value corresponding to the original (Point 3 of Figure 11). This demonstrates that the disappearance is due to the oxidation of MADE to NAD+,aa stated earlier. The enzyme appears to be specific for Nine. since the rate with NADPH as the substrate under identical con- ditiona is only 3% of that with man. Stoichiome the Reactior The balance study for the peroxidase reaction is shown in Table 5. 1.00? 0.80“ Point 1 5 3‘ - 1 c: g 0.60 ‘9 a .. ~ 9 0 g . n 0.40- h o 3 . re Point 3 0.20- \i \ db uh- Ii 1 j Time Figure 11 Illustration of the Stoiohiometry of the Peroxidase Reaction 56 Table 5. Stoichiometry for the peroxidase reaction. can ran—e. Y A 2.“- :n. HADH OxidiZed Ratio 32°: ”01“ moles szoz/uana 4.5 x 10'3 5.1 x 10'3 0.9/1 -3 -8 2.7 x 10 2.9 x 10 0.93/1 fl “5 he stated above it was shown that unDH was con- + verted to RAD . Therefore. these results demonstrate that the peroxidase reaction may be formulated as shown in Equation (1). + + 2;:th + H + 11202 :— HAD 4» 2320 (1) Spectrum of Enzyme The visible portion of the spectrum of peroxidase is shown in Figure 12. The enzyme gives a spectrum similar to that of FAD in regard to the maximum at 450 mu. There is no 370 mu peak. only a shoulder. Also of interest is the broad absorption band from 525 mu to 600 mu. Dolin reported (23) the appearance of a band from 520 to 600 mu when the peroxidase from g. faeoalis is reduced by either substrate or hydrosulfite. There is no explanation at present concerning the shoulders on both sizes of the 450 mu peak. There are no apparent heme absorption bands. IHET. .JHF. \ oudoanonom on» Ho annuoonm ma onmuam 3 Alfv namoodo>ms one cow one 000 one cow own .. ._...o 1' N00 1 n6 eoueqaos qv 58 ngactore Heat denaturation and acid precipitation doea not appear to release the flavin from the protein. It was also found that the presence of FAD. FMN or riboflavin in cofactor amounts has no appreciable effect upon the rate of the peroxidase. It is of interest that the flavin from the g. aecalis peroxidase (23) can be released quantitatively by heat denaturation at 1000 for ten minutes or by acid precipitation of the protein. It was identified as FAD. Also. Lightbown and Hogut reported (26) that the oxidasa from g. aubgilis required FAD. Receptors Various electron acceptors have been tried. as shown in Table 6. .Methylene blue. ferricyanide and substrate- lovol concentrations of FAD were found to approximately double the rate obtained without any acceptors present: but the rates with these acceptors are only one-seventh of that obtained with hydrogen peroxide. Benzoquinone and indophenol were found to be completely inactive. It would seem quite likely that the stimulation of the rate caused by the various acceptors in Table 6 is due to stimulation of the oxidase. The stimulation in rate 59 Table 6. Electron acceptors for peroxidase. _.. A. __.A w _, fl Concentration Rate Accept°r (Molarity) unite/0.05 ml enzyme None ""'"""' 0 o 9 a o 4 s x 10"4 18 2 2 ’ ran 1 x 10"4 3 Ferricyanide 5 x 10"4 2.1 Methylene Blue 1 x 10"4 2.5 Benzoquinone 5 x 10.4 0.3 Indophenol l x 10-4 0.5 __.__._ _‘ Standard Peroxidase Assay - RADB.concentration I 1.3 x 10’4M. Enzyme was the peak fractions from DEAR- celluloae, diluted 1 to 10. Protein concentration in diluted preparation: 0.094 mg/ml. obtained in the presence of these acceptors in by no means comparable with the rate of the peroxidase. and can readily be explained by the data to be presented on effects of alternate acceptors on oxidaae activity. Dolin reported(23) that FMN. FAD. methylene blue and indophenol did not function as oxidants for the‘g. fiaecalig peroxidase. He found menadione and l.4—naphthoquinone functioned as oxidants. The solubility was a big prOblem in our studies. Menadione was found to have a very slow rate at the highest levels we could achieve (on the order of 10-51%!) . 60 Inhibitor; For the inhibition studies the standard peroxidase assay was used. except that the buffer. IADH. enzyme and inhibitor (1 x l0~3MJ were preincubated for ten minutes at room temperature. The reaction was started by addition of hydrogen peroxide. It was found that amytal. EDTA and cyanide were ineffective. while p-hydroxymercuribenzoate inhibited 100% and N—ethylmaleimide approximately 50%. The peroxidase from g. feecalie is inhibited 20%.by p-hydroxymercuribenzoate and is completely inhibited by heavy metal ions. It is difficult to conclude if eulfhydryl inhibition in the mechanism of the heavy metal ions. Our studies would seem to implicate ~83 groups. Stabilit The effects of heating the enzyme at different temperatures are shown in Table 7. Table 7. Beat stability of the peroxidase. WA' 3* “L w “ “‘— Treatment % of Original Activity 40°C for 10 minutes 100 50°C for 5 minutes 100 60°C for '5 minutes 80 70°C for 5 minutes 0 61 Proggrtigg Q; the Ogidase Reconstitution of Pctivity __'—n: It was found that upon storage the oxidaao lost activity. Hadkler.§_ “l. reported (47) that the oxidase from.§. gaecalie lost enzymatic activity upon storage. They found that activity could be restored by addition of a variety of thiols together with FAD. Table 8 demonstrates that similar results are found when this oxidase in pre- incubated with cysteine and FAD for ten minutes at 37°C. Table 8. Reconstitution studios. Rate 3 R°c°n5titut‘°n Mixture units/0.l ml reconat. oxidaae Enzyme 1.5 Enzyme. cysteine and FAD 9.0 Enzyme. and cysteine 2.5 Enzyme and FAD 3.7 Enzyme. cyatcine and PM 3.5 The reconstitution is relatively specific for FAD. since reconstitution.with FMN gives only a fraction of the rate obtained with Fa . The oxidase from g. aghtilig requires FMN (26): that from g. faecalig requires FAD (47). 62 Pcccptcra The acceptors were tried anaerobically with recon— etitutod enzyme. (It should be noted that the excess flavin and cysteine were not removed. The results were corrected for non~enzymatic rate in the presence of FAD and cysteine in corresponding amounts.) It wax found that ENE at l x lO-4M and ferricyanide at 5 x 10-4n‘were ineffective as electron acceptors. In addition to molecular oxygen. the NEDH oxidaee catalyzed the reduction of excess FAD or methylene blue. Methylene blue appears to be an excellent acceptor even before reconstitution. The NADH oxidase from g. gcecelis has similar acceptor properties. Methylene blue was not tried in this case. Euennekens.gg.§;. reported (12) that the KARE oxidase isolated from pig heart could use methylene blue in cofactor amounts. but could not utilize FAD. (It should be noted that no reconstitution experiments were carried out by this group.) gnhibitore The inhibition studies were carried out on the enzyme before reconstitution using 5 x lO‘SM.methylene :éilll 63 blue as the electron acceptor. The preincubation of the 3Min each case). NADH and buffer enzyme. inhibitor (1 x 10“ was carried out for five minutes at room temperature. Cyanide. N-ethylmaleimide. p-hydroxymercuribenzoate and EDTA had no significant effect upon the rate. Heckler l. (47) found flADH oxidaae from g. faegalis was in- w “a. as. sensitive to iodoacetate. EDTA and cyanide. but was inhibited approximately 60% by p—hydroxymercuribenzoate. ihzennekenl 5;, 9.1;. (12) found 100% inhibition with p-hydroxymercuribenzoate. 22% with iodoaobenzoate and 19% with cyanide (l x 10-4M). Thus there appear to be some differences among the enzymes to the typical thiol inhibitors. gtcbility The effects of heating the enzyme at different temperatures are shown in Table 9. Table 9. Heat stability of the peroxidase. Treatment ‘% of Original Activity 40°C for 10 minutes 100 50°C for 5 minutes 100 60°C for 5 minutes 45 70°C for 5 minutes 2 SUMMARY The diaphorase from Lactobillug gage; has been isolated and purified approximately ninety-fold. It has been shown to be free from NADH oxidase and NRDH peroxidase activity. The diaphorase appears to be specific for NADH. It requires FMN for maximal activity: with an apparent Km for we of 1.8 x 10'6M. 0f the various electron acceptors tested. p—benzo- quinone and indophenol gave the highest rates. followed by ferricyanide. The diaphoraae activity is not appreciably affected by hydrogen peroxide. cyanide. EDTA or N—ethylmaleimide. The molecular weight appears to be in the range of 30.000 to 70;000. The properties of this diaphorase are in good agree- ment with those of the diaphorase isolated from g. faecalis by Dolin (24). There are. however. several major dif- ferencea between the prepertiea of this diaphorase and those of the classical diaphoraae of Straub (18): first. Straub's enzyme required FAD as cofactor and second. 64 65 methylene blue was an exCellent acceptor in his system. Neither is true of the g, gaggi.diaphorase. It was found that the peroxidase and oxidase activities could not be separated by ammonium sulfate fractionation. Sephadex GblOO and 64200 filtration. DEAE- cellulose chromatography. heat denaturation or calcium phosphate gel adsorption. Even though separation of these activities could not be accomplished. their properties could be studied indepen- dently. due to differences in their pH optima.- with a number of acceptors tried the stimulation was double. that without acceptor but it was found to be only twenty percent of that with hydrogen peroxide. Since these rates are not comparable with that of peroxide it is probable that they are stimulating the slight oxidase activity present at this pH. The peroxidase was completely inhibited by p~hydroxy~ mercuribenzoate and only 50%»with Neathyl maleimide. EDTA. cyanide and amytal were found to be ineffective. It‘wal found that the oxidaee lost activity during the isolation steps. The majority of the activity could be restored by preincubation with cysteine and FAD. (Nbckler reported (47) the same phenomenon with the oxidaae 66 from g. faecalis.) 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