3. S. Furuyama, D. Mayes, and C. Nugent, Steroids, 16, 415 (1970). 4. B.F. Erlanger, F. Borek, S. M. Beiser, et al., J. Biol. Chem., 228, 713 (1957). 5. J.J. Pratt, T. Wiegman, R. Lappohn, et al,, Clin. Chlm. Acta, 59~ 337 (1975), 6. W. Habl, F. Stahl, M. Buchner, et al., Endokrinologie, 68, 283 ~977), 7. G. Hobe, F. Barrels, and K. Schubert, Endokrinologie, 67, 282 (1976),

DETERMINATION OF THE u-GLYCOL GROUP IN NUCLEIC ACID COMPONENTS.

VII. AMPLIFICATION METHODS FOR THE TITRIMETRIC AND SPECTROPHOTOMETRIC

DETERMINATION OF TOTAL NUCLEOTIDES IN PREPARATIONS OF NICOTINAMIDE

ADENINE DINUCLEOTIDE USING PERIODATE OXIDATION

V. Zacharans, L.L. Yavarkovska, and A. I. Busev

UDC 547.963.32:543.86

Nicotinamide adenine dinucleotide (NAD) is the oxidized form of the redox coenzyme [i, 2], in which the pyrldine ring of nlcotinamide is capable of a reversible redox reaction, One of the most important coenzymes, NAD is present in all living cells and acts as the co- factor for dehydrogenases that catalyze redox processes in all biological systems [3]. It is widely used in biochemical research [3] and is produced in considerable and increasing amounts by the majority of biochemical companies [4].

A 5W method has been used for the determination of NAD [4-6], based on the measurement of its strong absorption in the 259-260 nm region due to the adenine and pyrldlne rings. This method is extremely sensitive but the effects of a large number of factors [7], due mainly to nucleotide contaminants which absorb in the same region of the spectrum, have to be taken into account. One single problem--the extinction coefficient of NAD--has been the subject of a good deal of work, of which the most important are [6, 8-12], The UV method gives the sum of 8- and u-NAD and the nucleotide contaminants that have similar spectral pa- rameters [13]. The spectrophotometric determination of NAD as its cyanide complex [6] gives the sum of ~- and u-NAD and the pyridine contaminants.

The enymatically active form of the coenzyme is 8-NAD alone, in which both nitrogen bas- es are linked by a S-N-glycosidic linkage with the C x atoms of D-rlbose. The isomer with the u-N-glycosidic linkage with D-ribose in the pyridine ring of nlcotlnamlde (u-NAD) does not display enzymatic activity. The 8-NAD content can be determined enzymatically [5].

o II

~ G-~ Z

I HO OH O NH z

H O - P r I

HO OH

There are no accurate methods for the determination of the content of NAD in its prep- arations. Difficulties arise because crystalline NAD preparations contain different amounts of water of crystallization, depending on the method of isolation, drying, and storage [14]. Our intention in the work described here was to develop an accurate tltrimetrlc method for

M.V. Lomonosov Moscow University. Translated from Khimlko-Farmatsevtlcheskii Zhurnal, Vol. 13, No. 4, pp. 102-107, April, 1979. Original article submitted July i0, 1978,

0091-150X/79/1304-0433507.50 �9 1980 Plenum Publishing Corporation 433

for determining the content of NAD in its preparations. This procedure, in conjunction with extant methods for the detection, determination, and separation of possible contaminants, including inhibitors of enzyme reactions [7, 15], could ha extremely suitable for the qual- ity control of NAD preparations, notably for the accurate determination of the total content of ~- and ~-NAD and nucleotide contaminants, and consequently of the water content, which is not unimportant in view of the high cost of the preparation,

The titrimetric version of the amplification method using perlodate oxidation, as sug- gested earlier for the determination of ribonucleosides and nucleoside 51~phosphates [1611 seems most promising. We set out to assess the potential of this method for further develop~ ment by examining whether the spectrophotometric determination of NAD can be carried out in this way [17]. Periodate oxidation has been used in the immobilization of NAD [18],

Stoichiometry of the Periodate Oxidation of NAD. The literature has nothing on the stoichiometry of the periodate oxidation of NAD. Consequently, we first examined the linear section of the plot of periodate consumption against the oxidation time of NAD. We used two methods of oxidation, differing in the mode of preparation of the perlodate solution, and three different excesses of the oxidant (Table i}. We found that with afive- or tenfold molar excess of periodate, regardless of the oxidation procedure, roughly 95% of the NAD is oxidized in the first 3 min , after which the periodate consumption slowly increases until the NAD is quantitatively oxidized, which takes place only after 2-4 h, depending on the excess of the oxidant and the oxidation procedure. We would have expected the quantitative oxida- tlon of NAD to be complete within a few minutes, as in the case of nucleoside 5'-phosphates [19], since even with a fivefold molar excess of perlodate there is a 2.5-fold excess of the oxidant over each cis-~-glycol group in the ~-D-ribofuranose parts of the NAD molecule. This distinctive feature of the periodate oxidation of NAD is evidently due to steric hindrance and/or to the molecular association of NAD molecules in aqueous solution as a result of in- teraction between the adenine and pyridine rings [20, 21]. The isomer distribution in the NAD preparations may also have some effect.

The oxidation of NADwith a 5-20-fold molar excess of periodate is rigorously stoichio- metric over a period of several hours and depends mainly on the oxidation procedure. When NAD is oxidized with a solution of potassium perlodate in dilute sulfuric acid ~H 0,8-1.6) it is more stable to ovaroxldation than when a solution of sodium periodate in water (pH about 3.5) is used. Thus the periodate oxidation of NAD shows the same general features as that of nucleoside 5'-phosphates [19]. However, since 0.01-0.005 M solutions of NAD (as the free acid) in water have pH 3.0-3.1, the oxidation of NADwith a solution of sodium periodate in water takes place at rather lower pH than that of nucleoside 5'-phosphates, Sincesolutions of NAD in water are most stable at pH 2.0-7.0 [22, 23] and since a 0.05 M solution of sodium periodate can be prepared by simply dissolving a weighed quantity of the preparation in water) NAD is more conveniently determined by oxidation with a 10-20-fold molar excess of the oxidant over a period of 3-5 h. When a 0.05 M solution of potassium periodate in i N sulfuric acid is used as oxidant the sulfuric acid has to be neutralized with alkali solution to pH 3.0 at the end of oxidation, before the necessary excess of molybdate is added,

Titrimetric Method for the Determination of NAD. Each NAD molecule contains two cis-~- glycol groups and consequently reacts with two periodate ions to form two iodate ions. We can represent the periodate oxidation as

NAD + 2 1 0 ~ > NAD =Te~aaldehyde+210~+2H,O.

At t h e end o f t h e o x i d a t i o n t h e e x c e s s p e r i o d a t e i s masked w i t h e x c e s s m o l y b d a t e a t pH 3 .0 a s 6 - m o l y b d o p e r i o d a t e and o n l y t h e 103 - fo rmed i n t h i s r e a c t i o n r e a c t s w i t h i o d i d e :

IO~ + 51- + 6H+ ___+ 31~ + 3H~O,

l i b e r a t i n g a q u a n t i t y o f i o d i n e e q u i v a l e n t t o t h e o x i d i z e d q u a n t i t y o f NAD, t i t r a t i o n o f wh ich consumes e x a c t l y 12 e q u i v a l e n t s o f sod ium t h i o s u l f a t e . Thus we can r e p r e s e n t t h e t i t r i m e t r i c v e r s i o n o f o u r p r o p o s e d method f o r t h e d e t e r m i n a t i o n o f NAD a s :

-I NAD m 210~ m 6Is ~ 12I m 12SsO~--,

i.e., one NAD molecule corresponds in the end-polnt determination to 12 equivalents of the titrant. The proposed method for the determination of NAD is one of the so-called ampllfica-

434

TABLE I. Stoichiometry of the Periodate Oxidation (n) of NAD as a F~nction of Oxidation Time and pH with Various Molar Ratios

Cperiodat e @ole/liter)/CNA D (mole/liter) = k at 18~

Oxidant

0.02 M potassium periodatein'0.1 Nsul- furic acid

0.025 M sodium perio- date in water 0.1 N

0.025 M potassium periodate in 0.1 N sulfuric acid

0.025 M sodium perio- date in water

0.050 M potassium periodate in 1 N suifurtc acid

0.050 M sodium perio- date in water

5

5

10

10

20

20

pH

1,6

3,4

1,4

3,5

Oxidation ]

time, dur- ~ }after 24-h ing which ]after 16-h :0.002:n=2.000 h i [ [~x~d"t*~ " ~ " I ~176

4--8

3--5

3 - 8

3--5

0,8

3,6

3--7

2--5

2,002

2,007

2,005

2,008

2,007

2,008

2,003

2,009

2,006

2,010

2,008

2,012

Note. When k was 5, i0 ml of 0.005 M NAD solution were oxi- dized with i0 ml of periodate solution; when k was i0 or 20, 5 ml of 0.01 M NAD solution were oxidized with 20 ml of per- iodate solution.

tion methods* [24-26]. The results of a titrimetric determination of NAD are summarized in Table 2.

We found that i ml of 0.025 N sodium thiosulfate solution corresponds to 1.3822 mg of N~. Because of the high molecular weight of NAP the equivalent weight in the possible acid-- base titration procedures will be several times greater. Since the periodate oxidation of NAD is rigorously stoichiometric and the final iodometric titration is highly accurate, the proposed amplification method has notably high precision (the relative standard deviation does not exceed 0.5%), and can be used for the determination of NAD and its preparations and also in some cases for its detection.

Spectrophotometric Method for the Determination of NAP. To assess the capabilities of the spectr0photometric determination of NAD using perlodate oxidation we followed our earlier work on nucleoside 5'-phosphates [17]. We first established that with a 25-50-fold molar ex- cess of periodate the oxidation of NAD proceeds to completion within i h and that increase in the oxidation time to 2 h does not cause overconsumption of the oxidant, The faster oxida- tion of NAD in this case is evidently due to the possibility of using larger excesses of the oxidant than in the titrimetric version and to the lower association of NAD in more dilute aqueous solutions.

We found that in 0.I M potassium iodide solution the absorption spectrum of the triiodide derived after periodate oxidation of NAD and amplification resembles those of the triiodlde derived after periodate oxidation and amplification of ribonucleosides and nucleoslde 5'-phos~ phates [17] and of triiodide in 0.I M potassium iodide solution.

We can represent the spectrophotometric version of the amplification method for the de- termination of NAP as;

I NAD m 210~- --- 6I z ---- 6I 3 .

For quantification we denote the molar extinction coefficient of NAD after periodate oxida- tion and amplification as e~AD. Here ga results when one molecule (or ion) of the test sub-

stance ultimately gives a molecules (or ions) of the substance responsible for: the analytical effect; a is the amplification number. Thus,

*We coined the term "amplifikatsionnyi metod" in [16] as a paraphrase of the English term "amplification method~" which is difficult to translate into Russian.

435

e=NAD= a.e,

where r is the molar extinction coefficient of the substance (or ion) whose absorption is measured in the end-point determination. Since in the case of NAD G =~^n6 and r is 26,000

liter mole-* c/n-* at 352 nm in 0.1 M potassium iodide solution, thenr =~ should be 156~000

liter mole-*cm-*. The average lvalueof r NAD derivedfrom ten measurements was 156,000 • 2500 liter mole-* cm-* at352 nm in 0,1 M 9otasslum iodide solution, The relative standard deviation::did not exceed 4%,i.e., the accuracyof the proposed method is typical of spectro c photometric determinations.

The molar extinction coefficient of NAD at its absorption maximum, 259 nm, does not ex- ceed 18,000 liter mole-* cm-* [4, 6-12]. In our proposed method the absorption of an equiva- lent amount of triiodide is measured at 352 nm, thus increasing the sensitivify of the de- termination of NAD by a factor of almost nine. After periodate oxidation and amplification, NAD can he determined with the same sensitivity as at the absorption maximum of 259 nm in the visible region of the spectrum at ~ k 400 nm, where a photoelectric colorimeter with the ap- propriate light filter can be used for the measurements of optical density,

EXPERIMENTAL

The equipment was described earlier [16, 17].

Nicotinamide Adenine Dinucleotide. The NAD preparation (free acid) was supplied by Re- anal (Hungary), catalog number 14136; C2,H27NTO,4P2; molecular weight 663.45. The $-NAD con- tent was certified as not less than 85% enzymatically and the water content as not more than 10%. Before use the 8-NAD content was checked enzymatically -- by reduction with yeast alcohol dehydrogenase (KF i.i.i.i) and ethanol by the procedure of [5] and found to be 84 • 2%. The total content of ~- and s-NAD, calculated from the molar extinction coefficient at 259 nm and pH 7.0, was 98 • 2%. The molar extinction coefficient was 18,000 liter mole-* cm -I [4], All NAD solutions were freshly prepared by dissolving a weighedquantity of the preparation in distilled water.

Reagents and Their Preparation. The 0.025 and 0.05 M sodium periodate solutions were prepared by dissolving a weighed quantity of the purified preparation in distilled water, The preparation of 0.025 M potassium periodate solution has been described earlier [16], The 0.05 M potassium periodate solution was prepared by dissolving with heating a weighed quanti- ty of the preparation (pure for analysis grade) in 1 N sulfuric acid, If the sulfuric acid concentration was lower some of the periodate precipitated after cooling. The periodate solu- tions were stored in dark glass bottles, in which they were stable for at least six months.

The other solutions and their preparation and storage have been described in earlier work [16, 17].

The oxidation of NAD was carried out at 18~ in a place protected from direct sunlight.

Procedure for the Titrimetric Determinations, The procedure used for the titrimetric determinations has been described earlier [16]. In the case of NAD either a sample weighed to • mg and containing approximately 0.05 mg-eq (normally 30-35 mg) or an aliquot of an aqueous solution equal to 5.00 ml and containing the same amount of NAD was used, Oxidation was carried out with 0.05 M sodium periodate solution (20 ml) over a period of 3-5 h. Be- cause of the increase in the excess of perlodate being masked the quantity of added molybdate was doubled.

The NAD content (x, %) was calculated from the equation:

x = (v~ - V ~ . N . M 6.n.g .1001

where V, i s t h e volume of t he s t a n d a r d s o l u t i o n of sodium t h i o s u l f a t e consumed i n t h e a n a l y t i - c a l t i t r a t i o n (ml) ; V2 i s t h e volume of t h e s t a n d a r d s o l u t i o n of sodium t h i o s u l f a t e consumed in t h e b l ank t i t r a t i o n (ml) ; N i s t h e n o r m a l i t y o f t h e s t a n d a r d s o l u t i o n o f : s o d i u m : t h i o s u l f a t e ( g - e q / l i t e r ) ; M i s t he m o l e c u l a r we igh t of NAD; g i s t he weigh t of NAD ( m g ) u s e d ; and n i s t h e number of moles o f p e r i o d a t e r e a c t i n g w i t h 1 mole of NAD (n = 2 ) ,

436

TABLE 2. Titrimetric Determination of NAD

Oxidant

Re la rive Mean Istandard

n result, ideviation,

0.025 M potassium periodate in 0.1 N sulfuric acid

0.025 M sodittm periodate in water

0.050 M potassium periodate in 1 N sulfuric acid

0.050 M sodium periodate in water

10 15

10 15

20 10

20 10

99,0

99,1

99,0

99,0

0,5

0,5

0.5

0,4

Procedure for the Spectrophotometric Determinations, The procedure used for the spectro- photometric determinations has been described earlier [17], To a 2.0 ~ 10 -5 M NAD solution (i-i0 ml) in 50 ml graduated flasks was added 0.025 M potassium periodate solution (2 ml) in 0.I N sulfuric acid (or 0.025 M sodium periodate solution). After 1-2 h oxidation the solu- tions were treated as described in [17]. The UV absorption spectra were recorded in l-cm quartz cells against the blank solution.

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