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

IV. COLORIMETRIC METHOD FOR THE DETERMINATION OF RIBONUCLEOSIDES

AND NUCLEOSIDE 5'-PHOSPHATES

A. I. Busev and V. Zaharans UDC 615.31:547.963.32].074:543.432

In the spectrophotometrlc determination of rlbonucleosides (I) and nucleoside St-phos- phates (II) and their absorbance in aqueous solution is measured at the absorption maximum in the 250-280 nm region, so that their content can be calculated from the molar extinction coefficient, and at 250, 260, 280, and 290 rim, so that the optical denslties D25o/D26o, D28o/D2~o and D2,e/D2so can be used to establish their purity with respect to other nucleic acid bases [1-2], However, the corresponding 2'-deoxyribonucleosides (III) and nucleoside 2'(3')-phosphates (IV) interfere in the determination of I and II. Extant colorimetrlc re- actions for RNA and DNA components are not sufficiently selective [3, 4].

When perlodate oxidation methods are used, III and IV do not interfere with the deter- mination of I and II. Various groups have suggested for the spectrophotometric determination of I and II and carbohydrates by periodate oxidation measurement of the reduction in absorb- ance of the periodate ion at the absorptlonmaxlmumat 223 nm [5-9], To eliminate interference from iodate and the oxidation products of carbohydrates and to simplify the experimental pro~ cedure Ikenaka has proposed a procedure whereby measurements can be carried out at 290 ran [10]. However, this version of the method cannot be used for the determination of I and II because they absorb strongly in this region of the spectrum. Kim has also suggested measure- ment at 232 nm of the quantity of iodate stoichlometrlcally equivalent to the number of oxi- dized ~-glycol groups after separation of periodate and iodate on anlon-exchange resin [ii]. We have previously put forward an amplification method for the tltrimetric determination of I and II based on the titrimetrlc determination with sodium thiosulfate of the quantity of 12 formed from oxidation and amplification of I and II [12]. Here we describe a colorlmetric version of this method for the determination of I and II, The quantity of iodine formed is determined as the trilodide resulting from the reaction:

I,+I- Z I? The equilibrium constant for this reaction at 20~ is only 1214 • 5 [13], i,e,, the re~

action is reversible. Complete displacement of the equilibrium to the right demands a suffi- cient excess of iodide. We found that the absorbance of I~ in the 350 nm region has the same

�9 %max as that of the 12 solution. However, increase in the iodide concentration is accompanied by increase in the intensity of the I~ absorption band in the 350 nm region, obviously because -- I~ > -- I, emax emax at the same wavelengths. We selected a KI concentration such that final concen-

tration is 0.i M. This iodide concentration gives reproducible optical densities of the tri- iodide solution and a linear calibration curve for iodate. Oxidation of one molecule of I or II forms one IO~ ion, which by reaction with iodide gives three molecules of Iz, which with excess iodide form three I~ ions, or, as is conventional in the presentation of amplifica- tion methods [14], 1 1 or II m i 10T E 31= ~ 31~,

We have found the molar extinction coefficient of I~, derived from purified KIO~ [15], to be 78,000 • 1000 for 0,1 M KI solution, in good agreement with the published molar extinc- tion coefficient of IF in 0.1 M KI solution at 352 nm of 26,000 [16]. In our method the mo- lar extinction coefficient of I~ is the same for all I and II regardless of the nature of the heterocyclic base and the number of phosphate residues. The molar extinction coefficient of I and II does not exceed i0,000 (pyrlmidine series) or 15,000-16,000 (purlne series) [1-2]. Thus with measurement at 352 nm our proposed method for the determination of I and II is five to ten times more sensitive than the conventional L~ spectroscopic method.

Our proposed method for the determination of I and II has previously been used for the detection of iodate in the presence of perlodate and vic-dlols (the resulting iodine is detec-

M, V. Lomonosov Moscow University. Translated from Khimiko-Farmatsevtlcheskii Zhurnal Vol, ii, No. 10, pp, 135-140, October, 1977. Original article submitted March 15, 1977,

0091-150X/77/III0,1427507.50 �9 1978 Plenum Publishing Corporation 1427

ted by reaction with starch [17]), for the spectrophotometric determination of vic~diels [16], of tartrate in the presence of citrate [18], and of iodide by periodate oxidation[19], and for the automated determination of iodate in carbohydrate mixtures after periodate oxida~ tion [20].

For the oxidation of I and II we tested KIO~ in 0.i N H2S04, which results in oxidation at pH 2.0-3.0~ and aqueous Nal04, which results in oxidationinweakly acidic or neutral sot lution at pH 4.0-7.0. We found that oxidation with KIO~ in 0,I N sulfuric acid is more suitable, since overconsumption of the oxidant does not occur, In neutral solution, espe- cially in the case of II, overconsumption of the oxidant even affects the result of the de~ termination. In 0.i N sulfuric acid oxidation of I and !I with periodate is complete within 2-3 min. Increase in the oxidation time to 3-5 h does not affect the result of the deter~ mination.

Figure 1 shows the absorption spectra resulting from oxidation of adenosine with I~ against a blank and of the components of the buffer and masking solutions before and after mixing against distilled water, The absorption spectra of I~ solutions derived from the oxidation of I and II resemble the absorption spectra of iodine in aqueous s~lutlons of KI, Solutions of pure preparations Qf I and !!, iodateand periodate, and of the components of the buffer and masking solutions do not absorb at wavelengths above 350 nm, However, addi- tion of Na=Mo04 solution to the buffer solution of C~CH2COOH and NaOH forms a compound that absorbs in the 350 nm region (though this does not affect the masking of periodate by molyb- date). Consequently, ablank run has to be carried out for each series of runs, This is also necessitated hy the fact that periodate commonly contains traces of iodate~ removal of which is difficult. Traces of iodate are also farmed when perlodate solutions are stored for. long periods. However, accurate recording of the quantity and concentration of all components of the buffer and masking solutions in the blank run eliminates all these influ~ ences,

We used a FEK N-57 photoelectric colorimeter to construct the calibration curves and to determine I and II. Beer's law holds for I and II in the respective concentration range 0.i-i.0 and 1.0-7.0 ~mole per 50 ml. Figure 2 shows the calibration curves for the determin- ation of disodium adenosine 5'-triphosphate. Calibration curves derived from measurements with a spectrophotometer pass through the origin of the coordinate system. Those derived with the FEK N-57 are displaced slightly upwards from the ordinate axis, which is obviously caused by the monmonochromaticity of the light produced with filters. Consequently, in this case concentrations of I and II below 0.i and 1.0 ~mole per 50 ml are undesirable in work with filters Nos. 1 and 2 respectively, since these give optical densities of less than 0.i. Our results for the colorimetric determination of I and II after statistical treatment in accordance with IUPAC recommendations are summarized in Table i.

The possibility of using an independent standard (KIOs) to construct calibration curves suggests another means of using this method for the determination of I and If. The slope of the calibration curve in the coordinates optical density-content of I or II in ~mole per 50 ml is directly proportional to the percentage content of I or II. Consequently, this method yields the content of I and II without the need to use standard solutions of I and II. The resulting correlation between the slope of the calibration curve and the percentage content of I and II is shown in Fig. 3. We determined the percentage content of I and II by the ti- trimetric version of the amplification method [12].

EXPERIMENTAL

Equipment. Absorption spectra were recorded with a Pye Unicam SP 8000 spectrophotometer (Great Britain). Construction of the calibration curves and determination of I and II were carried out with a FEK N-57 photoelectric colorimeter, with filters No. I (%elf = 360 nm) and No. 2 (%eff = 41q nm). All measurements used i cm cells.

Reagents and Preparation. We used commercial chromatographically pure preparations of I and II of varied provenance, containing various quantities of water. Before use their pur- ity was verified by UV spectroscopy [1-2] and their content by the titrimetric version of the amplification method [12].

We have described the preparation and storage of 0.025 M periodate solution from KIO~ in [12], The buffer solution was prepared by adding concentrated NaOH solution to 1 M CICH2- COOH solution to pH 2.9-3.0. The IM molybdate was prepared from Na2MoO~,2H20 "ch.d.a." ["pure

1428

TABLE i. Colorimetric Determination of Ribonucleosides and Nucleoside 5'-Phosphates after Periodate Oxidation and Ampli- fication as the Triiodide

[ 2 3 4 5

6

7

B 9

I0

II

12 13 [4

15

16 ~7

18

19

20 21

22

23

Compound

KIOs 5-ethylufidiae 5-bromourioine

Molecu- lar weight

214,00 272,20 323,11

Relative standard de- viation (S~),

% n

A B

10 1,2 7 3.5 7 3,0

Uridine Disodium uridine 5'-monophosphate

Disodium uridine 5'-diphosphate

Tr i so~um u~dine 5'-triphosphate

Cytidtne Cytid/ne 5'-monophosphate THsodium cytidine 5'-diphosphate

Trtsodium cytidine 5'-triphosphate

Adenosine Adenosine 5 ' - monophosphate Trisodium adenosine 5'-diphosphate

Disodium adenosine 5'-triphospham 4HzO

Inosine Disodium inosine 5'-monophosphate

D~odium inosine 5'-diphosphate

Disodium inosine 5'-triphosphate

Ouanosine Disodium guanosine 5'-monophos-

phate Disodium guanosine 5"-diphosphate

Disodlum guanosine 5'-triphosphate

244,20

368,15

448,13

550,09 243 20 323,20

447,16

549,13 267,24 347,23

493,15

623 23 268,23

392,19

472,15

552,14 283,25

407,19

486, L6

567,15

6

7

7 6 7

7

7 8 5

6

10 8

6

5

6 7

7

7

1

~ z j

0.142 ] 99,9 0,117] 81,4 0.141 ] 99,9 0.138 I 97,6 3,2

3,6 0.114 I 79,2

3,3 0,124 j 87,0

2,8 0,128 ] 90,0 2,6 0.137 ~ 97,4 2,3 0.100 I 68,8

3,4 0,133 I 93,6

3,6 0,120 I 84,0 2,6 0,139 ] 98,2 3,1 0,132 I 92,5

2,9 0,125I 87,5

2,5 0,138 I 97,7 3,0 0,135 [ 95,5

3,4 0.104 t 71,1

3,5 0,113 J 78,4

3,4 0.116 I 80,8 3,t 0.109 I 76,0

3,3 0.I12 ] 78,2

2,9 0.124 ] 87,2 i i

2,7 i0,126 88,1

Note. A) Filter No. 1 (0.i-i.0 Bmole I or 11/50 ml); B) Filter No. 2 (1.0-7.0 ~mole I or 11/50 ml).

1429

D

0,7

0,6

2;t. \ ...:,

3+50 400 45Ohm

D

o,~ 0,7 o,G O,~ 0,4 o,a o,z O,f"

t goE o,14o

b 0,130

a 0,120

0,I/0

Fig. i Fig. 2

Fig. i. Absorption spectra: a) IF

!

y+ ! r T r f I I r I t T r

a O, fO, Z O, a~O,~aGO,7OBOBm I,~ j,2 , i , i ~ 0 , I 0 0 r J z

b I f 3 " 4 "6" G 70 BO 90 100%

F i g . 3

from oxidation Of adenosine with periodate, Cadenosin e = 1.077,10 -5 mole/liter; b) 1 Msolution of Na=MoO4,2H=O in water; c) buf-

fer solution from i M CICH=COOH and NaOH with pH 3.0; d) solution prepared from the buffer solution and molybdate solution (see text).

Fig. 2. Calibration curves for the determination of disodium adenosine 5'-triphos- phate tetrahydrate: a) FEK N-57, filter No. I (keff = 360 nm); b) FEK N-57, filter No. 2 (~eff = 413 nm).

Fig. 3. Correlation between the slope of the calibration curves and the percentage content of I and II (FEK N-57, filter No. 2). The numbering of the points is that of Table i,

for analysis"] grade and the 0.5 M potassium iodide solution from "ch.d.a."grade Kland stored in a dark-glass bottle. The 0.0250 N (0.00417 M) standard KI03 solution was prepared from the purified compound [15].

Determination of I and II. Two procedures can be used for the determination of I and II: In the first the calibration curve is constructed from standard solutions of I or II and in the second from the standard KIO~ solution.

The standard solutions of I or II (0-15 ml) containing 0.i-i.0 Bmole (filter No. i) or 1.0-7,0 Bmole (filter No. 2) in 50 ml graduated flasks were made up to 20 ml with periodate solution (2.0 ml portions) and distilled water (2.0 ml portions), The solutions were then incubated for 15 min for oxidation in a~lace~protected from direct exposure to light. Buf- fer solution (5 ml portions) was added to the solutions, which were stirred; this was fol- lowed by addition of molybdate solution (2 ml portions) and careful stirring, and finally KI solution (I0 ml portions). After stirring the solutions were made up to 50 ml with dis- tilled water and then carefully stirred. The blank run was carried out similarly, After a few minutes the optical density was measured against the blank with the appropriate filter.

Use Of the standard KIO3 solution for construction of the calibration curve proceeds similarly, after the starting solution has been accurately diluted to one-twentieth the orig- inal concentration,

The molar extinction coefficient of I~ of 78,000 • i000 can be used when optical densities are measured with a spectrophotometer. The coefficient can also be established beforehand at 352 nm using carefully purified KIO= [15] and the KI stock solution.

LITERATURE CITED

i. D, Voet, W. B. Gratzer, R. A. Cox, and P. Dotyj Biopolymers, ~, 193-208 (1963). 2. E. Fredericq, A. Oth, and F. Fontaine, J. Mol. Biol., ~, 11-17 (1961). 3, H. N, Munro and A. Fleck, Methods Biochem. Anal., 14, 113-176 (1966). 4. G, D. Berdyshev, in: Modern Virology [in Russian], Kiev (1969), pp, 169-194, 5. J. S. Dixon and D. Lipkin, Anal. Chem., 26, 1092-1093 (1954),

1430

6. G. O. Aspinall and R. J. Ferrier, Chem. Ind. (London), No. 36, 1216 (1957). 7. R. Bergkvist and A. Deutsch, Acta Chem. Scand., 8, 1880-1888 (1954). 8. R. Bergkvist and A. Deutsch, Acta Chem. Scand., 8, 1889-1897 (1954). 9. L. I. Linevich, G. D. Erygin, and E. R. Ambartsumyan, in: Methods of Modern Biochem-

istry [in Russian], Moscow (1975), pp. 139-141. i0. T. Ikenaka, J. Biochem. (Tokyo), 54, 328-333 (1963). ii. D. K. Kim, in: Methods in Carbohydrate Research [in Russian], Moscow (1975), pp. 73-77. 12. A. I. Busev, V. Zaharans, and U. Mikstais, Khim.-Farm. Zh., ii, No. 3, 128-132 (1977) 13. E. N. Rengevich and E. A. Shilov, Ukr. Khim. Zh., Zh., No. 9, 1080-1086 (1962). 14. J. De Oliveira Meditsch, Rev. Quim. Ind. (Rio de Janeiro), 4~5, No. 531, 6~8 (1976). 15. Yu. V. Karyakin and I. l. Angelov, Pure Chemical Compounds. Handbook on the Prepara-

tion of Inorganic Reagents and Preparations under Laboratory Conditions [in Russian], Moscow (1974), p. 130.

16. G. Nisli and A. Townshend, Talanta, 15, 1377-1384 (1968). 17. G. Nisli and A. Townshend, Talanta, 15, 411-413 (1968). 18. G. Nisli and A. Townshend, Talanta, 15, 1480-1483 (1968), 19. R. Belcher~ J, W. Hamya, and A. Townshend, Chim. Analyt. (Bucharest), ~, 23-25 (1971);

Chem. Abstr., 75, 14603j (1971). 20. S. A. Barker, P. V. Peplow, and P, J, Somers, Carbohydr, Res,, 22, 201-204 (1972).

APPLICATION OF SPECTROSCOPIC METHODS TO THE INVESTIGATION OF HEPARIN

AND ACCOMPANYING IMPURITIES

V. P. Panov, V. V. Kobyakov, V. I. Svergun, V. M. Tsarenkov, and T. N. Birul'chik

UDC 615.273.53.011.17,074:543.42

Heparin performs a number of functions in the living organism, and one of the most im- portant is the ability to prevent blood clotting. As a natural anticoagulant, heparin is finding wide use in medicine, and the need for it continuously increases. The basic raw ma- terial for heparin production is cattle lung, as well as the mucous membrane of pig small in~ testine. Heparin is an acid mucopolysaccharide and is constructed from alternating D-glucos- amine and L-iduronic acid residues. It is characterized by a high content of sulfate groups (an average of five sulfate groups per tetrasaccharide residue [i, 2]), by the presence of sulfamine groups, and by a negligible content, or according to the latest data [3], total absence of N-acetyl groups. In vivo the bulk of the heparin is in an inactive form in a covalently bound complex with a protein carrier [4, 5], The breakdown of heparin--protein complexes is an important stage in any method of extraction of heparin. Subsequent separa- tion and purification of heparin provide for its liberation from accompanying compounds: li- pids, proteins, nucleic acids, mucopolysaccharides. The solution of the technological problems associated with the search for optimum ways to separate heparin from these substances while preserving its native structure would be unthinkable without the enlistment of modern physi- cal and physicochemical methods of investigation (identification of compounds, determination of the composition of their mixtures, establishment of the interrelationship of the structure of heparin macromolecules to their biological activity).

In this work we discuss the possibilities of using methods of UV, IR, and PMR spectros- copy for the detection o~ proteins, nucleic acids, and impurity mucopolysaccharides at indiv- idual stages or operations of isolation of heparin.

EXPERIMENTAL

Heparin samples from individual stages of the process and certain individual components of the mixtures were identified in the form of solutions in H20 and D20 of various concentra-

All~Union Scientific-Research Institute of the Technology of Blood Replacement and Hor- monal Preparations, Moscow. Moscow Endocrine Preparations Factory, Translated from Khimiko- Farmatsevticheskii Zhurnalo Vol. ii, No. i0, pp. 140-144, October, 1977. Original article submitted February 22, 1977.

0091-150X/77/III0-1431507.50 �9 1978 Plenum Publishing Corporation 1431