Carbonyl Additiou to Nicotinamide Adenine Dinucleotide in Frozen Solution

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 239, No. 9, September 1964 Printed in U.S.A. Carbonyl Additiou to Nicotinamide Adenine Dinucleotide in Frozen Solution SPECTRAL, FLUOROMETRIC, AND OPTICAL ROTATOR\- PROPERTIES OF THE OXIDIZED ACETONE ADDUCT M. I. DOLIN AND K. BRUCE JACOBSON From the Biology Division, Oak Ridge National Laboratory,* Oak Ridge, Tennessee (Received for publication, February 6, 1964) During an investigation into the hydrogen peroxide content of reduced nicotinamide adenine dinucleotide solutions, it was found that storing weakly alkaline solutions of commercial nicotinamide adenine dinucleotide at -20 led to the formation of five highly fluorescent modification products (1, 2). Hydrogen peroxide formation accompanied this modification reaction. Two of the fluorescent compounds formed in frozen solutions of commercial NAD are the reduced and oxidized forms of NADacetone (2); the acetone necessary for the synthesis is present as a contaminant in the commercial NAD. The spectra of the other three products suggested that they might be unidentified addition compounds of NAD. It was also shown that a variety of carbonyl compounds would add much more efficiently to NAD in frozen solution (2) than in liquid solution (3). The adducts formed (analogues of reduced NAD) are slowly oxidized at - 20 to analogues of oxidized NAD. Interest in these addition reactions stemmed from several considerations. First, because fluorometry is widely used to assay reduced pyridine nucleotide (4), it seemed necessary to define the conditions under which the highly fluorescent adducts formed and to investigate these compounds further. Second, there was a possibility that the adducts formed in frozen solution might resemble the pyridine nucleotide modification products that arise either spontaneously (5) or in the presence of various enzyme systems (6, 7). Relevant to the latter point is the suggestion that carbonyl adducts of NAD may be intermediates in hydrogen transport (8). After pure oxidized NADacetone had been prepared, interest shifted to the properties of this compound, because it is a good model for studying dinucleotide conformation. Evidence will be presented that the folded dinucleotide conformation of oxidized NAD-acetone strongly affects the spectral, fluorometric, and optical rotatory properties of the compound. Preliminary reports have dealt with certain phases of this work (1, 2). EXPERIMENTAL Materials PROCEDURE Synthesis of Modification Products at -20 -For large scale experiments, 0.5 g of commercial NAD was dissolved in 125 ml * Operated by Union Carbide Corporation for the United States Atomic Energy Commission. of water, the solution was chilled to 0, and the ph was brought to 11.9 by adding 1 N KOH. Equal volumes of the solution were then transferred into two glass centrifuge bottles (loo-ml capacity) which were placed in a deep freeze at -20. Soon after the solution freezes, the bottom portion turns yellow and on further storage the entire solution becomes yellow. The fluorescence intensity is maximal after 3 days of storage. At this time the solutions have an orange tint; on prolonged storage they become deep orange. After 3 days of storage, the solutions were thawed and heated to 55 for 15 minutes to destroy most of the remaining NAD. This heating does not appreciably change the concentration of oxidized NAD-acetone. The yield of modification products in this procedure is given in Table I. For preparing smaller amounts of modification products, the above procedure can be scaled down loo-fold, with no detectable change in the results. Synthesis of NAD-Acetone at Room Temperature-Reduced and oxidized NAD-acetone were also prepared according to the method of Burton, San Pietro, and Kaplan (10). The oxidized acetone adducts, prepared either at -20 or at room temperature, were isolated in pure form on columns of DEAE-cellulose (see below). The eluates containing pure compound were lyophilized, and LiCl was extracted from the residue at 0 with a 1: 1 mixture of ether and ethyl alcohol. The suspension was kept at 0 for 3 to 1 hour with occasional stirring, after which the residue was centrifuged from the solution at 0 and the supernatant fluid was discarded. This procedure was repeated three more times, and the final residue was washed with ether and dried under vacuum. The final product was a yellow powder. Preparation of Oxidized NMN-Acetone-Oxidized NAD-acetone was hydrolyzed with snake venom diesterase (11). After the hydrolysis was completed, as judged by the fluorescence increase (Table III), the reaction mixture was placed in a waterwashed dialysis bag and dialyzed against five changes of distilled water (10 volumes each time) at 4 to separate the protein from the nucleotide. The dialysate was chromatographed on a DEAE-cellulose column (see below) and a lithium salt was prepared, as already described for the dinucleotide. Based on the dinucleotide used, the pure, yellow lithium salt was obtained in 90 % yield. Preparation of a-nmn-a small amount of CX-NMN was prepared by hydrolysis of wnad (1.4 pmoles) with venom diesterase (11). The NMN moiety was isolated on a sheet of 3007

2 3008 Oxidized NAD-Acetone Vol. 239, No. 9 TABLE I Synthesis of modijkation products from nicotinamide adenine dinucleotide Modification products were synthesized in l-ml volumes, under the conditions given in Methods. NAD was determined with alcohol dehydrogenase (9). Concentrations are expressed as mole fraction of initial NAD. - NAD Pabst Lot 330 preparation storage temperature -20" 4._ Initial acetone ANAD - I Total adduct formeda Reduced Oxidized Total Reduced I NAD-acetone Oxidized formed* Total 0.10 (acetone-free) c " p-nad (acetone-free) with added acetone 0 Estimated as NAD-acetone. * Determined on eluates from DEAE-cellulose sheets (Fig. 2). c Unidentified compound, estimated as NAD-acetone i - DEAE-cellulose and eluted from the sheet with 0.02 M Trischloride, ph 7.5. Other Products-p-NAD were purchased from Sigma Chemical Company and Pabst Laboratories. These compounds contained 0.25 to 0.35 mole of acetone per mole of pyridine nucleotide (2). Acetone-free P-NAD was prepared as previously described (2). NMN and adenosine diphosphate ribose were obtained from Pabst laboratories, and (r-nad (Lot 60 B-696) was obtained from Sigma Chemical Company. DEAEcellulose sheets (designated DE-20) were purchased from H. Reeve Angel and Company, Inc.; DEAE-cellulose for columns was purchased from Brown Company and from Eastman Organic Chemicals. Snake venom diesterase, with no detectable 5 -phosphatase activity, was obtained from Worthington Biochemical Corporation. Methods Chromatography on DEAE-cellulose Sheets-DEAE-cellulose sheets were converted to the chloride form as previously described (12). Just before use, a sheet of DEAE-cellulose (chloride form) was washed again with water for one-half to twothirds of its length in a descending chromatography apparatus. The compounds were then spotted on the wet sheet, which was developed for about 3 hours by descending chromatography with a buffer mixture of 0.02 M Tris-Cl, ph 7.5, containing 0.1 M NaCl. The sheets were then dried in air. Chromatography on DEAE-cellulose Columns-DEAE-cellulose was suspended in water, and the smaller particles were removed by decantation several times. The column was poured to final dimensions of 3.8 x 40 cm and was washed successively with 2 liters each of 0.1 N NaOH, water, and 0.1 N HCl, and then with 4 liters of water. Reaction mixtures were diluted to 2 pmoles of dinucleotide per ml, adjusted to ph 8.5, and added to the column. After a small ultraviolet-absorbing fraction was eluted with water, elution with 0.05 M LiCl at 4 ml per minute resulted in appearance of oxidized NAD-acetone in the eluate at 550 to 1000 ml of LiCl (Fig. 3). Eluates were checked for the following ratios: absorbance at 260 rnp to absorbance at 360 mp (ph 7.2), fluorescence at ph 7.2 to absorbance at 360 ml (ph 7.2), and fluorescence at ph 12 to fluorescence at ph 7.2. Fractions in which the ratios deviated from constancy were discarded. Rechromatography of the first half of the dinucleotide peak (see Results ) was necessary. For chromatography of the diesterase-hydrolyzed NAD-acetone, it was necessary to wash the column with water much more extensively than was necessary for the dinucleotide. The mononucleotide could not be adsorbed to the DEAE-cellulose until effluents from the water washing reached a ph greater than 4. The mononucleotide was eluted with 0.04 M LiCl. Eluates were monitored as described for the dinucleotide. The mononucleotide was pure after one passage through the column. The AMP, derived from diesterase hydrolysis of the dinucleotide, was eluted with 0.2 M LiCl. Lithium salts of the AMP and oxidized NMN-acetone were prepared as described for the dinucleotide. Determination of Pyridine Nucleotides-The following molar absorptivities were used: /3-NAD, in 1 M KCN, 5.9 X lo3 at 325 rnp (13); P-NMN, in 1 M KCN, 6.2 X lo3 at 325 mp (14); fi-nadh, 6.22 x lo3 at 340 rnp (13); reduced NAD-acetone, 6.28 X lo3 at 340 rnp (ph 12). This value was obtained on aliquots taken from a solution containing 5 mm P-NAD, 4 to 6 M acetone, and 0.02 M KOH. Adduct formation in this solution was maximal after incubation for 2 hours at room temperature, after which time the acetone adduct began to be oxidized, as evidenced by an increase in the absorbance at 400 mp. The oxidized forms of NAD-acetone and NMN-acetone were determined by use of the molar absorptivities listed in Table II. In a mixture containing both oxidized and reduced NAD-acetone, the difference between the 340 rnp absorbance at ph 7 and ph 1.5 allows an estimate of the reduced compound present (3), whereas the absorbance of the 360 rnp band at ph 1.5 or the 400 rnp band at ph 12 allows the determination of the oxidized compound. Both (r-nad and a-nmn were determined by using the molar absorptivity quoted by Sigma Chemical Company for the cyanide adduct of a-nad, Lot 60 B-696 (5.4 x 103 at 332 mp). Where was also assayed with yeast alcohol dehydrogenase (9). Pentose Determination-Determination of pentose was performed by the orcinol procedure described by Dische (16). Organic Phosphate Determination-The method of Sumner (17) was used to determine phosphate after perchloric acid oxidation (18). Fluorometry-Fluorescence was measured in a Farrand tluo-

3 September 1964 M. I. Dolin and K. B. Jacobson 3009 TABLE II Spectrophotometric constants of oxidized NAD-acetone and oxidized NMN-acetone Spectra were determined in 0.01 N HCl, 0.02 M potassium phosphate buffer, ph 7.2, and 0.01 N KOH. The ph values were checked with a glass electrode before and after the spectra were measured. The molar absorptivity values are based on the measurement of the organic phosphate content of the acetone adducts, as described in Methods. Compound PH Xrn~X I Amar 3 NAD-acetone (oxidized) m c x 10-s +w c x 10-a f x 10-a 11.4 NMN-acetone (oxidized) NAD-acetone (oxidized) + diesterasea Sum of AMP+ + NMN-acetone (oxidized) a Enzymic hydrolysis carried out as described in Methods. b The l value for AMP at 255 rnp, ph 7.2, is 14.6 X lo3 as calculated from Reference 15. rometer, with thiamine filters. The primary filter had its maximum transmittance at 362 rnp (half-band width, 34 mp). The secondary filter had its maximum transmittance at 490 mp; 50 y. of the maximum transmittance was obtained at 434 and 560 mp. Under the conditions used, fluorescence intensity was directly proportional to concentration of the fluorescent substance. Optical Rotation-Rotations were determined in a Rudolph high precision polarimeter which was kept at 22 in a constant temperature room. The polarimeter tubes used were 2 dm long and had a capacity of 1 ml. Spectropho2ometry-Spectra were taken with a Beckman model DU spectrophotometer, equipped with 3-ml absorption cells (l-cm light path). Spectra of eluates from DEAE-cellulose paper were sometimes taken in 0.2-ml microcells (l-cm light path). The fluorescence of oxidized NAD-acetone does not interfere with the spectral measurements, since no change in the absorbance is detected when the distance from the absorption cell to the photomultiplier is changed by a factor of 2 (Beckman DK2 spectrophotometer). The strong fluorescence of oxidized NMN-acetone causes some interference as shown by the fact that the measured absorbance is low by 2.5yo at an absorbance of 1.2 and by 4.5% at an absorbance of 2.0. Measurements on the mononucleotide were made in the region in which absorbance was directly proportional to concentration. RESULTS Fluorescent Modi&ation Products Formed in Frozen Solution Spectral and Chromatographic Properties-As described previously (2), NAD, 5 mm in frozen solution at ph 11.9, is converted to at least five fluorescent, colored derivatives, two of which result from reaction with the acetone which commonly contaminates commercial NAD (0.25 to 0.35 mole per mole of NAD). Fig. 1 presents the spectra of solutions of commercial NAD and also of acetone-free NAD, after the modification products have formed (see Methods ). The yellow-orange solution formed from commercial NAD shows a band at 340 rnp which disappears partially in acid; this decreased absorption in O.* 1 A f 300 3bo 380 4io io 460 WAVE LENGTH (mfl) FIG. 1. Spectra of NAD solutions after 3 days of storage at -2O, ph A, NAD, Pabst Lot 330, containing 0.34 mole of acetone per mole of NAD; 1:30 dilution of frozen solution. B, acetone-free b-nad (2); 1:30 dilution of frozen solution. 0, potassium phosphate buffer, 0.02 M, ph 7.2; A, HCl, PH 1.4; 0, KOH, ph 12. acid is accompanied by the formation of a new band at about 290 mp. This behavior suggests the presence of a dihydropyridine (3). The presence of another compound or compounds is indicated by the existence of an absorption band at 360 rnp (ph 1.4), which shifts to 400 rnb at ph 12. Such bands are typical of oxidized carbonyl adducts of NAD (2, 10). By contrast, the orange solution obtained from acetone-free NAD (Fig. 1B) seems, from the almost total disappearance of the 340 rnp band in acid, to contain mainly a dihyropyridine. Chromatography of the alkaline solution of commercial NAD, after storage for 3 days at -2O, is shown in Fig. 2. The five fluorescent spots will be referred to as I to V in order of increasing Rp value. Spots I to III are orange in visible light and have a greenish fluorescence. Compounds IV and V are colorless at the concentrations shown; they have a blue-white iluorescence at neutral ph which changes to a yellow-green fluorescence at alkaline ph. The quenching spot above Spot IV is probably a mixture of ADP-ribose and AMP. It will be noted that the RR of Compound IV corresponds to that of NADH, and the RF of compound V, to NAD. Compounds IV and V have been identified as the reduced and oxidized forms of NAD- I B

4 3010 Oxidized NAD-Acetone Vol. 239, Tc o. 9 CHROMATOGRAPHY OF NAD MODIFICATION PRODUCTS ( DEAE- CELLULOSE) 0 n A 0 C DEFG NAD (ALK. -X)G) ~~K$TiNAthE NAD NADH ADPR AMP li!i Q FIG. 2. Chromatography of NAD modification products. Details are given in Methods. The NAD used was Pabst Lot 330 (Fig. 1). Cross-hatched areas fluorescent spots (Mineralight, are quenching spots; open areas are SL 2537 filter). The amounts of nucleotide placed on the origin were 1 pmole of the alkaline NAD (Track A), 0.8 pmole of nicotinamide, and 0.4 pmole of the other compounds. acetone (2). Briefly, the identification rests on the comparison of Compound V with oxidized NAD-acetone prepared in liquid solution ( Methods ) and then purified on a DEAE-cellulose column. Compound V and oxidized NAD-acetone have the same ultraviolet and visible spectra, molar fluorescence, molecular rotation, and mobility on DEAE-cellulose. Compound IV is autoxidizable in frozen solution (neutral or alkaline) or during the drying of a DEAE-cellulose sheet; the product of the reaction is oxidized NAD-acetone. The orange compounds with low RF values, I to III, presumably carry a higher negative charge than IV or V. These orange compounds have not been identified, but the spectra suggest that they may be mixtures of oxidized and reduced carbony1 adducts (most of the oxidation takes place during the drying of the chromatogram). The situation is complicated by the fact that orange compounds at the RF of I to III are also formed from acetone-free NAD. When the solution containing the modification products formed from acetone-free NAD is chromatographed, there is only a small amount of compound at Spot IV, and no detectable compound at Spot V, but the orange spots, I to III, are present. The spectra of eluates from these spots resemble those of mixtures of oxidized and reduced carbony1 adducts of NAD (2, 3); that is, there is an acid-labile compound with a band at 340 rnp and an acid-stable compound with a peak at 380 to 385 rnp. The latter shifts 40 rnp toward the visible end of the spectrum at ph 12. Since the solution used for chromatography seems to contain mainly reduced pyridine nucleotide (Fig. lb), the inference is that the modification reaction results in the formation of reduced adducts and that the latter become partially oxidized during the chromatographic procedure. The modification product formed in strong alkali has spectral (19, 20), fluorometric (19) (Table III), and chromatographic properties that are quite different from those of the compounds discussed above. On DEAE-cellulose, the mobility of the strong alkali product is slightly lower than that of NMN and equal to that of oxidized NMN-acetone. Table I shows the yield of modification products from commercial NAD, which contains acetone, and from acetone-free NAD. Total adduct concentration was calculated on the assumption that the absorbance at 340 rnp which disappears in acid represents a reduced adduct of NAD (3) and that the band at 360 rnp which remains in acid and the band at 400 rnp observed in alkali are attributable to an oxidized carbonyl adduct of NAD (2, 10). In the presence of 0.34 mole of acetone per mole of NAD, approximately half of the total product formed is NAD-acetone (the ratio of reduced to oxidized acetone adduct is 1.2). The remainder of the product is presumably accounted for by the orange compounds, which are also formed in the absence of acetone. As shown in the last line of Table I, a mixture of acetone-free NAD plus acetone produces results similar to those given by commercial NAD. The synthesis of comparable amounts of NAD-acetone in liquid solution would require a 150-fold excess of acetone over NAD (26 ). At 4, only small amounts of modification products are formed, in either the presence or absence of acetone. Following the discovery that NAD-acetone was efficiently synthesized in frozen solution, a variety of other carbonyl compounds were found to act as good nucleophils (2). With acetoin, acetaldehyde, and fructose, compounds giving fluorescent spots at positions IV and V are obtained in addition to fluorescent TABLE Molar fluorescence of modijication products derived from nicotinamide adenine dinucleotide Fluorescence was determined with a Farrand fluorometer (thiamine filters). Compound NAD-acetone (oxidized).... NMN-acetone (oxidized).... Alkali-hydrolyzed NAD-acetone dized)b... Strong alkali product of &NADc NADH... NAD-acetone (reduced)d.... III (oxi- L! Relative fluorescence per mole ph 7.2 ph 12 R a Ratio of fluorescence at ph 12 to fluorescence at ph 7.2. b Hydrolyzed for 30 minutes at 100 in 1 N KOH (see the text). c Prepared in 5 N KOH at 100 (19) and neutralized with HC101. Relative fluorescence is based on the initial concentration of NAD. Chromatography on DEAE-cellulose shows that there are four fluorescent products present. The product which makes the major contribution to the near ultraviolet spectrum of the 5 N KOH solution has a fluorescent ratio, R, of 44. d Not a pure compound (see discussion of Fig. 3).

5 September 1964 M. I. Dolin and K. B. Jacobson 3011 derivatives that migrate to the area of Spots I to III on chromatograms. The Spot V compounds of acetoin and acetaldehyde have spectra that are very similar to that of oxidized NADacetone; the spectrum of the Spot V compound obtained in the presence of fructose closely resembles that of the oxidized dihydroxyacetone adduct designated DPN-Dox I by Burton et al. (10). Ribose phosphate and ADP-ribose also act as nucleophils (or as sources of nucleophils), giving rise to orange compounds which migrate to the Spot I to III region of the chromatogram. Of the nucleophils tested, the compounds containing ribose were the only ones that enhanced the formation of orange derivatives. This suggests that the orange compounds formed from acetonefree NAD may be ribose adducts, the ribose-containing nucleophil arising through the well known alkaline cleavage reaction of NAD to ADP-ribose and nicotinamide (21). It does seem, however, that various neutral carbonyl compounds, in addition to causing the formation of compounds analogous to IV and V, will also support the formation of low RF compounds different in color and fluorescence from those produced from NAD alone or from NAD and ribose phosphate. This has not been investigated in detail. It is of interest that orange compounds similar in spectral and chromatographic properties to those derived from NAD (I to III) have also been found in frozen solutions of NADH. Conditions for Optimal Synthesis of Fluorescent Products-Since alkali extraction is routinely used (4) in the preparation of tissues for fluorometric analysis of NADH, it seemed worthwhile to define further the conditions under which fluorescent products are formed in alkaline solutions of NAD. These experiments were performed before any of the fluorescent modification products had been identified. Solutions of commercial NAD (5 mm) were brought to the desired ph with KOH and stored under three different temperature conditions: -20 for 2 days, 4 for 2 days, and 55 for 15 minutes. The extent of the modification reaction was determined by measuring the fluorescence intensity of the solution. The results will be expressed as relative fluorescence (measured at ph 7.2) per mole of initial NAD, on a scale in which NADH equals 100. For solutions stored at ph 10.5, 11.7, and 12.2, the results are: at 4, 0.26, 7.9, and 42; at 55, 0, 15, and 26; and at -2O, 15, 116, and 82. At -2O, the results are not appreciably altered either when the NAD concentration is increased from 5 to 25 pmoles per ml or when the solutions are saturated with O2 or Hz prior to the addition of alkali. The ratio of fluorescence intensity at ph 12 compared to ph 7.2 is approximately 1 for the solutions stored at 4 and 55, and approximately 1.8 for the solutions stored at -20. A ratio of 1 seems to be characteristic of reduced pyridine nucleotides (Table III), suggesting that a 4 or 55, the modification product remains primarily in the reduced form. This is also shown by the spectra of solutions stored at 4. The results summarized above indicate the extent to which NAD, in the presence of about 0.3 mole of a neutral carbonyl compound per mole of NAD, can interfere with the fluorometric assay of NADH. Appropriate controls, of course, would distinguish the products from NADH. The rest of this paper will be devoted to the properties of oxidized NAD-acetone. The properties of Compound V (oxidized NAD-acetone synthesized at -20 ) will be compared with those of the oxidized acetone adduct synthesized in liquid solution (10) in order to document the identity of the two compounds. Properties of Oxidized Acetone Adduct Preparation of Pure Oxidized NAD-Acetone (Compound V)- Pure oxidized NAD-acetone can be isolated on columns of DEAE-cellulose (see Methods ). In this procedure, as shown in Fig. 3, the absorbance at 360 rnp and the fluorescence are eluted in a constant ratio; however, the second half of the peak is contaminated with compounds absorbing at 260 rnp. Two of the contaminants are NAD and an unidentified yellow, nonfluorescent compound. Rechromatography of the first half of the peak yields a pure compound, as judged by the constancy of the absorbance to fluorescence ratios in the elution pattern. The chromatography of oxidized NAD-acetone prepared according to Burton et al. (10) is virtually identical with that of Compound V. Excess absorbance at 260 rnp and a nonfluorescent yellow compound begin to emerge with the second half of the fluorescence peak, so that preparation of a pure compound requires rechromatography. Bttempts to purify oxidized NADacetone on columns of Dowex l-cl were unsuccessful. The resin apparently catalyzes modification reactions which become more troublesome as the concentration of pyridine nucleotide placed on the column increases. Postulated Structure of Oxidized NAD-Acetone-The pure compound is a dextrorotatory pyridine nucleotide. Treatment of the compound with venom diesterase releases 1 mole of 5 -AMP and 1 mole of a fluorescent NMN analogue (the latter determined from its organic phosphate content). The AMP has the chromatographic and spectral (14) properties of 5 -AMP, serves as a substrate for adenylic deaminase (22), and, according to its optical rotation (Table IV), contains ribose in the /3 configuration. Therefore, the AMP moiety is unmodified 5 -AhlP. The fluorescent moiety, according to chromatographic and electrophoretic criteria, has the same net charge as NMN. Electro / d I/./ I 5io 590 6jO LiCl (ml) FIG. 3. Isolation of oxidized NAD-acetone on a DEAE-cellulose column (chloride form). 0, absorbance at 260 rnp, ph 7.2; 0, absorbance at 360 rnp, ph 7.2; a, fluorescence, normalized to absorbance at 360 rnp (ph 7.2) ; 0, absorbance at 260 rnw in excess of that attributable to oxidized NAD-acetone; n, absorbance at 260 rnp contributed by NAD (assayed withalcohol dehydrogenase).

6 3012 Oxidized NAD-Acetone Vol. 239, No. 9 TABLE IV Molecular rotation of nucleotides Aqueous solutions containing from 20 to 25 pmoles of the compound in question per ml were brought to the indicated ph with 1 N KOH. The ph was checked with a glass electrode before and after determination of the optical rotation. Sources of compounds: &NAD, Sigma Lot GlB-654 (containing 2.9 mole y. of a pyridine nucleotide resistant to Neurospora NADase) ; 8-NADH, Sigma Lot l-44; AMP, Pabst Lot 217. The acetone adducts were prepared as described in Methods. Values for 01- and 8-NMN and cr-nad were calculated from the data in Reference 30; the molecular weights of NMN and NAD were taken as 334 and @-NMN cu-nad ol-nmn AMP Compound AMPa NAD-acetone NAD-acetone Compound NMN-acetone V (reduced) (oxidized) (oxidized) PH a Prepared from NAD-acetone (oxidized). b Approximate value. -22,000-20,600-41,300-12,800 +9, ,400-17,800-19,200-18,800-30,000~ +26, , , ,400 +5, , ,800 phoresis in borate buffer shows that at least two vicinal hydroxyl groups are present. After treatment with a 5 -phosphatase, the fluorescent moiety behaves like a pyridinium ion and migrates to the cathode on electrophoresis at ph 7.0. When Compound V is incubated with a mixture of diesterase and 5 -phosphatase, 2 moles of Pi are released for every mole of adenine present in the original dinucleotide. According to the behavior summarized above, Compound V appears to be a typical pyridine dinucleotide, containing a fluorescent NMN moiety. Identification of the fluorescent dinucleotide as oxidized NADacetone (2) has been reviewed in a previous section. The postulated structure (10, 23) for oxidized NAD-acetone is shown in Fig. 4A, an enol form being shown in Fig. 4B. The pyridone ring is formed by the addition of acetone to position C-4 of NAD to give reduced NAD-acetone, followed by oxidation and a cyclization between the carbonyl group of acetone and the carboxamide group. Cyclization was postulated to explain the very weak amide test given by oxidized NAD-acetone (10, 23). This reaction resembles the synthesis of 2-pyridones through the cyclization of /3-ketoamides and ketones (24)..Mobcular Weight Determination-Support for the structure shown in Fig. 4 has come from determination of the molecular weight of the modified pyridine ring by mass spectrometry, according to Biemann and McCloskey (25). In separate experiments, ribose 5-phosphate, NMN, and the mononucleotide form of Compound V (NMN-acetone) were introduced into the ioniz- R CH3 C"3 A 0 FIG. 4. Proposed structure of oxidized NAD-acetone (R = adenosine diphosphate ribose). A, ph 7.2; B, ph (See footnote 2.) ing beam of a mass spectrometer under conditions that yielded positive ions. Both of the mononucleotides appeared to be cleaved at the nicotinamide ribosyl linkage with release of the pyridine ring as the highest molecular weight ion produced. Thus, one of the main peaks found for NMN had the molecular weight of nicotinamide, i.e The outstanding peak produced from oxidized NMN-acetone had the weight of the pyridine ring shown in Fig. 4, i.e. 160, with no activity detectable at a mass of 122. There was, in addition, a peak which would correspond with the demethylated derivative. None of the fragments produced from ribose 5-phosphate interfered with the interpretation of the mass spectra. Nuclear Magnetic Resonance-Preliminary examination of DzO solutions of oxidized NAD-acetone in an NMRi spectrometer (Varian, 60 megacycles) showed clearly the existence of a single methyl peak (relative area equivalent to 3 protons) at 3.65 p.p.m. versus internal water. This result is consistent with the suggestion that one of the methyl groups of acetone is the site of condensation with NAD.2 Elementary Analysis-Oxidized NMN-acetone, prepared as described in Methods, was redissolved in water (ph of solution, 7.2) to a concentration of 20 mg per ml, and the solution was centrifuged to remove fibers introduced by the DEAE-cellulose column. The clear solution was lyophilized, redissolved in water, and lyophilized again. According to elementary analysis, the preparation fits the following empirical formula. Calculated: C 36.8, H 4.38, N 6.13 (moisture 7.88) Found : C 36.97, H 4.66, N 6.39 (moisture 8.13) Moisture was determined on a sample dried to constant weight at 80 under vacuum. The presence of chloride was shown qualitatively. These analytical data are in complete agreement with the structure of Fig. 4 and the molecular weight data. 1 The abbreviation used is: NMR, nuclear magnetic resonance. 2 Results of NMR analysis of oxidized NMN-acetone sistent with the structure shown in Fig. 4B, but indicate are conthat at neutral ph the molecule Fig. 4A. The pyridone is a tautomer of the structure shown in ring would contain an -NH group, but no methylene group. Acetone addition takes place at the 4-position of the pyridine ring. The analyses, performed by Dr. 0. Jardetzky, will be presented in detail elsewhere. Recently, the addition of acetone to N-propylnicotinamide has also been shown by NMR spectroscopy to occur at the 4-position (J. Ludowieg, personal communication). R

7 September 1964 M. I. Dolin and K. B. Jacobson NAD-ACETONE -- NMN -ACETONE (ox) o DH il.9 compound. In a similar experiment, the pk, of the dinucleotide is found at 9.5. Fluorometric Properties of Oxidized Acetone Adduct-The fluerescence-ph curves of the mono- and dinucleotide forms of NADacetone are shown in Fig. 7. These results are strikingly similar to those obtained with FAD and flavin mononucleotide (27,28). With both the flavins and the oxidized acetone adducts, the following behavior is found. Between ph 5 and ph 7 the mononucleotide is much more fluorescent than the dinucleotide, the factor being approximately 10 for the flavins (27) and 13.5 to 14 for the oxidized acetone adducts. As the ph drops below 5, the fluorescence of the dinucleotides increases, and at ph 2 (ph 2.8 for the flavins), the fluorescence is maximal. At this n 3io 4bo WAVE LENGTH (m,u) FIG. 5. Spectra of the oxidized forms of NAD-acetone (--) and NMN-acetone (- - -). 0, potassium phosphate buffer, 0.02 M, ph 7.2; 0, KOH, ph Readings were taken at 2-rnp intervals in the region of absorption peaks and at lo-rnp intervals in the other spectral regions. Spectral Properties of Oxidized Acetone Adducts-The spectra of oxidized NMN-acetone and oxidized NAD-acetone are compared in Fig. 5. At neutral ph, both compounds have an absorption band at approximately 360 rnp, which shifts to approximately 400 rnp at ph 12. At the latter ph, both the mono- and dinucleotides have a less intense peak at 340 mp. The spectrum of the dinucleotide in 1 M KCN is identical with that found in KOH at ph 11 to 12. It will be noted that the 360 and 400 rnp bands of the mononucleotide are markedly shifted (6 to 7 mp) toward the ultraviolet region when compared with those of the dinucleotide. In addit ion, there is a large hyperchromic effect, which, at ph 7.2, amounts to 26 y0 at 255 rnp (Table II) and 16 y0 at 360 ml*. This degree of hyperchromicity seems to be the largest reported for a pyridine nucleotide (cj. References 13, 26). A blue shift, accompanied by hyperchromicity, can also be obtained with the dinucleotide. if the latter is brought to acid ph (not shown). At ph 2.1, both the positions and intensities of the absorption bands of the dinucleotide resemble those of the mononucleotide (Table II). This change is reversible. The spectrum of the mononucleotide is the same at ph 2.1 and 7.2. Table II lists the spectrophotometric constants of the monoand dinucleotides. As a check on the accuracy of the constants, it is seen that summation of the absorbances of AMP and oxidized NMN-acetone accounts quantitatively for the hyperchromicity found on hydrolysis of oxidized NAD-acetone with venom die&erase. The spectra and spectrophotometric constants discussed above are identical with those found for the mono- and dinucleotide forms of Compound V. Spectrophotometric titration of the mononucleotide is shown in Fig. 6. The pk,, as determined from the absorbance changes at either 240, 360, or 400 rnp, is 9.7. Along with the sharp isosbestic points, the fact that the same pk is found at three different wave lengths is good evidence for the purity of the WAVE LENGTH (mp) FIG. 6. Spectrophotometric titration of oxidized NMN-acetone. A standard volume of the compound was added to 3 ml of Tris-Cl, M. The buffer had previously been adjusted to the indicated ph with KOH; ph values were checked with a glass electrode before and after determination of the spectra. The absorbance changes illustrated are completely reversible FIG. 7. Comparison of fluorescence-ph curves of the oxidized forms of NAD-acetone (lower curve) and NMN-acetone (upper curve). q I, KOH; A, HCl; 0, potassium phosphate, 0.02 M; 0, sodium citrate, 0.05 M; n, sodium acetate, 0.1 M; A, glycine-hci or potassium glycinate, 0.05 M. 20

8 3014 Oxidized NAD-Acetone Vol. 239, No. 6 point the dinucleotides are 65 % as fluorescent as the mononucleotides. The apparent pk in this region is 3.3 for oxidized NAD-acetone and 3.5 for FAD (27). When HCl is used as the acid, the fluorescence of both the mono- and dinucleotides is quenched below ph 2 (ph 2.8 for the flavins). With the acetone adducts, quenching does not take place when H&GO4 is the acid used; in fact, the fluorescence is somewhat higher than anticipated. It is possible, therefore, that the quenching caused by HCI is attributable to Cl- and not to H+. In 0.5 N NaCI, the fluorescence of the dinucleotide (ph 5.5) and the mononucleotide (ph 6.7) forms of the oxidized acetone adduct is quenched by 20 and 25%, respectively. It is of interest that the pk, at 9.5 to 9.7 shows up in the ph-fluorescence curves of the acetone adducts. On the alkaline side of the pk, the fluorescence values of the mono- and dinucleotides are almost equal. Recently (29) a situation similar in some respects to that described above has been noted for 5-amino-NAD and its mononucleotide. At maximum, the mononucleotide was 2.3 times as fluorescent as the dinucleotide. In Table III, the molar fluorescence of NADH is compared with that of several NAD modification products. The significance of the spectral and fluorometric data presented in this and the preceding sections will be considered later in relation to the conformation of oxidized NAD-acetone (see Discussion ). Optical Rotation of Acetone Adducts-Oxidized NAD-acetone is dextrorotatory. This observation was of interest from the beginning of this study, because it seemed to offer a means of investigating the manner in which such analogues arise. The only other dextrorotatory pyridine nucleotide reported is (Y- NAD (30). The data of Table IV show that most of the dextrorotatory properties of oxidized NAD-acetone are attributable to some feature of the dinucleotide structure and not to the rotation of the individual moieties of the dinucleotide. In brief, reduced NAD-acetone, prepared from P-NAD according to Burton et al. (lo), is more strongly levorotatory than the initial pyridine nucleotide. When the reduced adduct is oxidized with ferricyanide (10) and then purified on a DEAE-cellulose column, the resultant product is dextrorotatory and has the same molecular rotation as Compound V. Oxidized NMN-acetone, however, is only weakly dextrorotatory at neutral ph. The rotation increases approximately 4.3-fold between ph 7.1 and Over a comparable ph range, the rotation of the dinucleotide increases only 1.2-fold. At ph 7.1 the sum of the molecular rotations of oxidized TABLE Pentose determination by orcinol reaction (AMP standard) Absorbance at 660 rnp was measured after 45 and 90 minutes of heating. fl-nmn, Pabst Lot 6001; a-nad, Sigma Lot 60 B-696; a-nmn prepared from the c~-nad as described in Methods. Compound V Moles of pentose per mole of pyridine nucleotide 45 min 90 min p-nad a.nad &NMiY cu-nmn NAD-acetone (oxidized) Compound V NMN-acetone (oxidized) TABLE Alkaline hydrolysis of oxidized NAD-acetone Hydrolysis was carried out in dim light. At the indicated times, aliquots were removed for determination of the fluorescence at ph 7.2 and ph 12 (see Methods ). KOH concentration Temperature VI Time Fluorescence remaining ph 1.2 ph 12 N min % % NMN-acetone plus AMP is -13,460, whereas the rotation of the dinucleotide at this ph is +25,600. Even at ph 10.4 to 10.7, the molecular rotation of the dinucleotide is 26,000 more positive than the sum of the rotations of the mononucleotide and AMP. The discrepancy, therefore, must be accounted for by the structure and conformation of the dinucleotide. This point will be amplified below (see Discussion ). The available data for (Y- and,&nmn (30) (Table IV) also indicate that the dinucleotides (01- or P-NAD) are more dextrorotatory than would be expected from the rotations of the individual moieties. This effect, however, is much smaller than for the acetone adducts. A final decision on this point must await the investigation of the possible ph dependence of rotation in the NMN series. Pentose Determination-In the orcinol determination (Table V), /3-NAD and fl-nmn give about the theoretical yield of ribose. Both ol-nad and oxidized NAD-acetone, however, are partially refractory in this procedure. As expected, the refractory pentose is the one attached to the pyridine ring. The inference to be drawn is that the strength of this N-ribosyl bond has been modified in the dextrorotatory analogues of NAD. Hydrolysis of Oxidized NAD-Acetone-The nicotinamide ribosyl bond of oxidized NAD-acetone is much more stable to alkaline hydrolysis than is the corresponding bond in &NAD (Table VI). After 2 hours of heating at 50 in 0.1 N KOH, there is no detectable change in the fluorometric properties of the oxidized acetone adduct. Under the same assayed fluorometrically in 5 N KOH (19), is 95% destroyed. At loo, in 1 N or 2 N KOH, oxidized NAD-acetone is converted to a chromophore that has only 15% of the initial fluorescence of the dinucleotide. The chromophore (which may be nictotinamide-acetone) has a peak at 300 rnp at ph 6.9. The peak shifts to 325 rnp at ph 12. On DEAE-cellulose paper, the RF of the chromophore is somewhat greater than that of nicotinamide. When oxidized NAD-acetone is heated in 0.1 N HCl at 50 for 2 hours, there is a 2% conversion of the dinucleotide to the mononucleotide. Heating in 2 N HCl at 100 for 45 minutes causes an almost stoichiometric conversion of the dinucleotide to the mononucleotide. Chromatography reveals the presence of two minor unident.ified components. Attempts to hydrolyze oxidized NAD-acetone enzymically have been unsuccessful. Neurospora NADase (31), used under conditions that would have caused the hydrolysis of 330 pmoles of &NAD, had no effect on the fluorometric or chromatographic properties of oxidized NAD-acetone. A particulate rabbit brain

9 September 1964 M. I. Dolin and K. B. Jacobson 3015 NADase (32) was also without effect on the oxidized acetone adduct. Presence of Oxidized NAD-Acetone in Pyridine Nucleotide Preparations Preformed oxidized NAD-acetone is present in small amounts in some commercial pyridine nucleotide preparations. (The compound was identified by its spectral, fluorometric, and chromatographic properties.) For illustration, the following concentrations, expressed as mole per cent of the total pyridine nucleotide, have been found in various preparations: ar-nad, Sigma Lot 60B-696, 2%; NADH, Pabst Lot 2208, ph 8.0, fresh solution, 0.64 $$ (after storage for 5 months at -2O, 1.0%); NADH, Pabst Lot 2215, ph 8.2, fresh solution, 0.4%. Various preparations of P-NAD obtained from Sigma Chemical Company or Pabst Laboratories contain from 0.02 to 0.04% oxidized NAD-acetone. DISCUSSION Carbonyl reaction with the pyridinium ring was described by Najjar, White, and Scott (33) and Huff (23). Subsequently Burton et al. (10, 34) synthesized a variety of carbonyl adducts of NAD and described the general properties of these compounds. All the condensations required large excesses of nucleophil. In contrast, the present work has shown that at -20 very small concentrations of nucleophil will react almost to completion with NAD. (This result may be caused by concentration of solutes in the liquid phase of the frozen solutions.) This finding has certain practical consequences. First, the ease with which the reactions take place may lead to spurious results in the fluorometric assay of natural materials for pyridine nucleotides. For instance, if homogenates were stored in the frozen state at alkaline ph, it is possible that naturally occurring carbonyl compounds would condense with NAD to form adducts. If the proper controls were not run, these adducm could be mistaken for NADH. Table III shows that the oxidized form of the adducts might be especially troublesome, oxidized NAD-acetone, for instance, being 5.4 times as fluorescent as NADH. It has been shown that the oxidized form of the adduct can form spontaneously through oxidation of the reduced form in frozen solution. On the other hand, the very fact that these addition reactions take place so efficiently at -20 makes possible the use of rare carbonyl compounds as nucleophils to carry out syntheses which might otherwise be impracticable. The ease with which the condensations take place suggests that pyridine nucleotide preparations may be contaminated with a variety of adducts. In fact, small amounts of oxidized NAD-acetone have been found in commercial pyridine nucleotide preparations, especially in NADH. As mentioned previously, storage of frozen NADH solutions causes the formation of orange compounds which seem to have the spectral characteristics of NAD adducts and which resemble the low RF orange compounds formed in frozen alkaline solutions of acetone-free P-NAD. In this regard, it may be mentioned that after oxidation (35) the inhibitor isolated from frozen NADH solutions (5) has the spectral properties (2, 10) expected of an oxidized carbonyl adduct. The spectrum differs from that of oxidized NAD-acetone, and previous work (34) had shown that neither reduced nor oxidized NAD-acetone was inhibitory to a variety of NAD-linked dehydrogenases. It has been suggested (8) that adducm of NAD may be inter- mediates in the mechanism of hydrogen transfer for certain pyridine nucleotide-linked dehydrogenases. Adduct formation with enzyme-bound NAD would certainly be expected, in view of the ease with which the condensation takes place with free NAD under appropriate conditions. Whether such enzymebound adducts occur as normal, kinetically active intermediates remains to be determined. The proposed structure for oxidized NAD-acetone (10, 23) (Fig. 4) is supported by the results of molecular weight determinations (mass spectrometry) and elementary analysis. Evidence for a pk, at 9.5 (dinucleotide) or 9.7 (mononucleotide) has been obtained from spectrophotometric (Fig. 6) and fluorometric (Fig. 7) titrations. These results may be explained by the tautomerism illustrated in Fig. 4. On the alkaline side of the pk, the compound would acquire 1 more net negative charge (as actually found by electrophoresis). In addition, at alkaline ph the ring system would be more fully conjugated, which is in agreement with the observed bathochromic shift of the absorption bands (Fig. 6). The most interesting properties of oxidized NAD-acetone relate to its conformation. The evidence presented strongly indicates the existence of a complex between the adenine and modified pyridine rings of oxidized NAD-acetone. This complex affects the spectral, fluorometric, and optical rotatory properties of the compound. Complexes between the ring systems of dinucleotides have previously been proposed for FAD (27, 28), NADH (36), and various NAD analogues (13, 29). Fluorometric evidence for complexing is presented in Fig. 6, which shows that oxidized N&IN-acetone is much more fiuorescent than the dinucleotide in the ph region 5 to 7. The very similar results obtained for FAD and 5avin mononucleotide (27, 28) have been interpreted by Weber (28) to indicate intramolecular complexing between the two ring systems of FAD, with resultant quenching of the fluorescence. The marked spectral shifts (Fig. 5) and hyperchromicity (Fig. 5 and Table II) shown by the oxidized acetone adducts also point to the existence of such a complex (13, 26). A pk, in the region 3.3 to 3.7 is found in the ph-fluorescence curves of FAD, 5-amino-NAD, and oxidized NAD-acetone. The increase in fluorescence in this region (on acidification) has been attributed (29) to protonation of the amino group of adenine with consequent disruption of the intramolecular complex. In the case of oxidized NAD-acetone, dissociation of the complex by acid is shown both by the 5uorometric data (Fig. 6) and by the fact that at ph 2.1 the spectrum of the dinucleotide markedly resembles that of the mononucleotide (Table II). The spectrum of the mononucleotide is the same at neutral and acid ph (cf. FAD (26)). One consequence of intramolecular complexing in the oxidized NAD-acetone molecule has not previously been reported for other dinucleotides. Table IV shows that the molecular rotation of oxidized NAD-acetone cannot be accounted for by the sum of the rotations of its constituent moieties. At neutral ph, the dinucleotide is approximately 40,000 more positive than expected. Two hypotheses may be offered to explain the optical rotatory properties of the dinucleotide. First, the dextrorotatory properties may be attributable to the folded conformation itself. A model is afforded by hexahelicene (37). Because of molecular overcrowding, this molecule exists in either of two helical conformations. Both isomers have been isolated and are strongly optically active ([cjd -3640, +3707). It is pertinent to note here that Dhe positive optical rotation of undena-

10 Oxidized NAD-Acetone Vol. 239, No. 9 tured nucleic acid is attributed to the helical conformation of the macromolecule (38). A second hypothesis derives from the work of Jardetzky and Jardetzky (39, 40), who have shown by NMR spectropscopy that the ribose rings of purine and pyrimidine ribosides have different conformations (39) which may be mirror images (40). Such conformations would be consistent with the observations that purine ribonucleosides are levorotatory, whereas pyrimidine ribonucleosides are dextrorotatory (41), and that pyrimidine ribonucleosides are refractory in the orcinol procedure (16). The dextrorotatory NMN analogues c+nmn and NMN-acetone are also refractory in this orcinol determination (Table V). It may be suggested, therefore, that on oxidation of reduced NAD-acetone, the ribose attached to the pyridine ring may undergo a change in conformation that affects both the optical rotation and the stability of the nicotinamide ribosyl linkage. At neutral ph, oxidized NMN-acetone is weakly dextrorotatory. The large increase in rotation as the ph is raised may reflect a conformation change in the ribose, a change that may be strongly enhanced in the folded dinucleotide structure of oxidized NAD-acetone. NMR analysis of the mono- and dinucleotides may provide an answer to this question. SUMMARY 1. At -2O, carbonyl addition to nicotinamide adenine dinucleotide takes place very efficiently. Small concentrations of carbonyl compounds will react almost to completion with NAD to form analogues of NADH. The latter are slowly oxidized at -20 to analogues of oxidized NAD. 2. Commercial preparations of NAD are contaminated with acetone (about 0.3 mole per mole of NAD); therefore, when such preparations are frozen at alkaline ph, a mixture of reduced and oxidized NAD-acetone is formed. In addition, three orange derivatives are synthesized, in either the presence or absence of acetone. These compounds have spectra similar to those of carbonyl adducts of NAD. Compounds resembling the orange modification products are also found in frozen solutions of NADH. 3. Oxidized NAD-acetone and its mononucleotide, oxidized NMN-acetone, have been prepared in pure form on columns of diethylaminoethyl cellulose. Comparison of the properties of the mono- and dinucleotides has indicated strongly that the dinucleotide exists in a folded conformation, because of complex formation between the adenine and pyridine rings. The complex strongly affects the spectral and fluorometric properties of the molecule, and is responsible for the fact that the dinucleotide is dextrorotatory. For instance, at neutral ph, the sum of the molecular rotations of oxidized NMN-acetone plus adenosine monophosphate is -13,460, whereas the molecular rotation of oxidized NAD-acetone is +25,600. Two hypotheses are considered to explain the optical activity of the dinucleotide: (a) the optical activity may be ascribed to the folded conformation itself, and (b) the ribose attached to the pyridine ring may undergo a conformation change because of the folded conformation of the molecule. Acknowledgments-We are indebted to Dr. D. G. Doherty for assistance with the polarimetric determinations, and to Drs. R. H. Dinius and 0. Jardetzky for determinating the nuclear magnetic resonance spectra of oxidized NAD-acetone. REFERENCES 1. DOLIN, M. I., Biochim. et Biophys. Acta, 63, 219 (1962). 2. DOLIN, M. I., AND JACOBSON, K. B., Biochem. and Biophys. Research Communs., 11, 102 (1963). 3. BURTON, R. M., AND KAPLAN, N. O., J. Biol. Chem., 206, 283 (1954). 4. LOWRY, 0. H., ROBERTS, N. R., AND KAPPHAHN, J. I., J. Biol. Chem., 224, 1047 (1957). 5. FAWCETT, C. P., CIOTTI, M. M., AND KAPLAN, N. O., Biochim. et Biophys. A&x, 64, 210 (1961). PURVIS. J. L., Biochim. et Biophus. 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