Spectrophotometric Analyses of 2-Deoxy-~-ribose in Presence of Nucleosides JOACHIM K. SEYDEL' and EDWARD R. GARRETT2 College of Pharmacy, Universify of Florida, Gainesville, Fla.
b An ultraviolet chromophore, A,,, 2 6 1 mp, unique to 2-deoxyribose is produced under well defined conditions of acid, temperature and timee.g., 5 hours a t 80" C. in 1.OM HCIand is a direct function of sugar concentration. This serves as a basis for a direct and sensitive assay of deoxyribose. The chromophore is generated by specific acid catalysis. The Arrhenius' parameters are given and the conditions for production are defined. In alkali, the chromophore i s shifted to 2 9 3 mp and vanishes by a specific hydroxyl ion catalyzed reaction process. This property serves as the basis for deoxyribose analysis in the presence of other stable chromophores. The chromophore is extractable b y chloroform and can b e spectrophotometrically analyzed in this solvent.
T
degradation of sugars by concentrated acids into products which have ultraviolet spectra a t 210400 mp has been used as the basis for the empirical correlation of spectrophotometric absorbance and Concentration (6, 7 , 12). These degraded sugars also react with specific reagents to produce colored compounds which can serve as a basis for analysis (1, 2 , 9, IS). The direct spectrophotometric analyses of degraded sugars in concentrated acids that are available are not sensitive to small concentrations and the resultant chromophores cannot readily discriminate among sugars (6, 7 , 11, 12). A classical procedure for the analysis of deoxyribose has been the method of Dische ( f , 2 )which is not truly specific to this sugar (10) and depends on the reaction of the products of acid degradation with diphenylamine. The present interest in nucleosides, nucleotides, and the nucleic acids demands the development of specific and sensitive assays for derived sugars that are operative in the absence and presence of sugar precursors ( 8 ) . When the hydrolyses of 5-iodo-2deoxyuridine (3) and deoxyuridine (4) under mild conditions of acid and temperature were followed by changes HE THERMAL
1 Present address, Borstel Research Institute, Borstel 2061, Germany. 2 To whom communications should be addressed
in ultraviolet spectra, higher absorbance readings were obtained than could be assigned to the uracil product. This phenomenon could be esplained by the transformation of 2-deoxyribose to a substance which has a strong chromophore at 261 mp as shown by curve ii in Figure 1. This chromophore could be satisfactorily produced under well-defined conditions of acid, temperature, and time so that it was a direct function of sugar concentration. The chromophore was extractable with chloroform and could be destroyed by treatment with mild alkali at room temperature (curves B and D of Figure 1). These properties should serve as a basis for a direct and sensitive spectrophotometric assay of deoxyribose even in the presence of other stable chromophores. EXPERIMENTAL
Apparatus. T h e Beckman Model D U ultraviolet spectrophotometer with 0.12 mm. slit width for acid readings and 0.15 mm. slit width for alkaline readings a n d the Cary Recording ultraviolet spectrophotometer, Model 15 were used in these studies. Materials. 2-Deoxy-~-ribose, A grade, Calbiochem. Deouyuridine, Uridine and Cracil, Nutritional Biochemicals Corp. All other chemicals were of analytical reagent grade.
=
[D]
+
[N]
+
[u] (1) where [ N ] represents the concentration of nucleoside and the e are the respective molar absorptivities and are 1300 for 2-deoxyribose, 8120 for uracil, 9950 for uridine, and 9900 for deoxyuridine. Dilute an aliquot of the acid degraded mixture 1 : l with 1.351 NaOH and maintain at room temperature for 24 hours. Read the absorbance a t 295 mp against a blank prepared in the same manner where [NaOH] = 0.15. Since the nucleosides do not absorb and the degraded deoxyribose chromophore is destroyed, twice the observed absorbance is A261
tDZ6'
6NZ6l
[VI
( 2) The is 5600. Reacidify the alkaline sample by diluting 1: 1 with 0.2.11 HCl and again read at 261 mp against a blank similarly treated where [HCl] = 0.025. Four times the observed absorbance is A295 = EU295
=
[N]
+
[VI
(3) It follows that the 2-deoxyribose A261'
ENz6'
€CZ6'
concentration to be assayed is
[ D ]= e7
(a4261
-
~~261')/eDZ6'
(4)
B
Direct Spectrophotometric Procedure for 2-Deoxyribose. Prepare the
calibration curve for 2-deoxyribose. Use 1.0 to 10.0 X 10-4.11 concentrations at 1.0 X intervals in 1.0.11 HC1. Maintain samples in thermostatted bath at 80.0' C. for no less than 5 hours with a maximum of 24 hours. Read the absorbance, A , at 261 mp against a 1.OA1 HCl blank and plot against 2deoxyribose concentration [ D ] as in Figure 2. For our conditions [ D ] = A/1300. Unknown concentrations of 2-deoxyribose must be diluted to the 1.0-10.0 X 10-4Ji range for assay of absorbance, A .
Direct Procedure in Presence of Uridine, Uracil, and Deoxyuridine.
Prepare appropriate two component mixtures of 2-deoxyribose, [ D ] , and either uracil, [C-1, uridine, or deoxyuridine, so t h a t the total absorbance after heating a t 80.0' C. in 1.11 HCl does not rxceed 1.00 a t 261 mp. If the total absorbance is in excess, dilute with known volumes of 1N HCl. The absorbance in acid solution at 261 mp is
Figure 1 . Ultraviolet spectra of final product of 0 . 2 5 M HCI degradation of 1 O-3M 2-deoxyribose, DR After 5 hours a t 80.0°, Read a t 5 X 10-4M DR and 0 . 2 5 M HCI E. In alkaline solution of A a t p H 12, diluted 1 : 1. Read a t 5 X 1 O-4M DR and 1 .OM N o O H . C. O n reacidification of E. Read a t 2.5 X 1 0-4M DR and 0.1 M HCI. 0 . After B stands for 1 3 minutes a t room temperature.
A.
VOL. 37, NO. 2 , FEBRUARY 1965
271
t
I
with chloroform to 10 ml. Read the absorbance, A , a t 261 mp against a chloroform blank similarly prepared. l'repare a calibration curve as in Figure 2. For our conditions [D]= A/516. Unknown concentrations of 2-deoxyribose must be diluted to the 1.0-20.0 X 10-4M range for assay of absorbance, A. Apply this method to nucleosidedeoxyribose mixtures and since the nucleoside is not extractable by chloroform under the stated conditions, the absorbance at 261 mp of the chloroform solution is directly proportional to the initial and derived deoxyribose concentrations. RESULTS AND DISCUSSION
Effects of Acidity and Temperature. An ultraviolet chromophore IO' [Deoxyriboss]
Figure 2. Direct proportionality between absorbance, A, a t 261 mp of degraded 2-deoxyribose (5-24 hours at 80.0" in 1N HCI) and concentration of 2-deoxyribose [D] A.
B.
Direct reading agalnst 1 N HCI blank where A = 1300 [D] Reading of combined 10 ml. chloroform extracts of 5 ml. aqueous solution agalnrt chloroform blank where A = 5 1 6 [ D ]
Calculate the concentrations of the nucleoside and uracil from Equations 2 and 3 if it is desired. I n those cases where the nucleoside is not stable under the conditions of the assay, where uracil is not present in the original mixture and where uracil and deoxyribose products result from the assay procedure, modify the calculations. Subtract the value of [GI obtained from Equation 2 from the [D] obtained from Equation 4 on the assumption of equimolar products to obtain the original 2-deoxyribose concentration of the assayed mixture. Chloroform Extraction Procedure.
Extract 5 i d . of the acid-degraded mixtures described previously for the direct spectrophotometric procedure two times with 3 nil. of CHC1, and collect the separated chloroform extracts. Dilute the combined extracts
'
(curve A of Figure 1) appears with time (Figure 3) on the thermal degradation of 2-deoxyribose in aqueous HC1 solutions. The asymptotic absorbance, A m, achieved with time was the same for 60" as for 80" C. The rate of appearance of the 261 mg chromophore is not strictly first order. The S-shaped curve that is apparent in Figure 3 is consistent with the premise that the 261 mp chromophore is associated with a final product of a sequential series of reactions which is most probably two. Prior to the appearance of the 261 mp chromophore a band, Amsx = 225 mp, of lessened absorptivity arises by an apparent first order process which is proportional to hydrogen ion activity, U H + , a t a constant temperature (Figure 4) where u H + = f[HCl], and the activity coefficient, f, is obtained from literature values ( 5 ) . Unfortunately, this 225 mp absorption band is not specific since at many sugars-eg, ribose-absorb this wavelength after degradation in acid solution. Whereas the 261 mp chromophore is highly extractable with chloroform, the 225 mp is not. The possibility exists that the compound responsible for the 225 mp chromophore may be a precursor to the compound responsible for the 261 mp chromophore since a slight decay in the absorbance of
I
t
.tlUU
Figure 3. Absorbance at 261 m,u os function of time and temperature from degradation of 1 O-3M 2-deoxyribose in 0 . 2 7 N HCI
10
272
20
40 TIME (HOURS)
30
ANALYTICAL CHEMISTRY
50
60
70
0
0.5
1.0
A n+
Figure 4. Linear dependence of apparent first order rate constants, k in seconds-' on hydrogen ion activity OH+ for 1 O-3M 2-deoxyribose A.
B.
Rote of appearance of 2 2 5 m p chromophore a t 60.0' Rate of appearance of 2 6 1 mpchromophore a t 80.0' after induction period.
the former is concomitant with the increase in absorbance of the latter. The rate of appearance of the 261 mp chromophore is proportional to the hydrogen ion activity, uH+, a t a constant temperature (Figure 4). The apparent first order rate constant, k in seconds-', for the appearance of the 225 mp absorbance can be obtained from a plot of the logarithm of the difference in absorbance, A, and the absorbance, A , , of the approach to a maximum value a t any time, tin seconds, in accordance with the expression log lA
- il = - (1%/2.303)1+ constant (5)
Similarly, the apparent first order rate constants for the achievement of the asymptotic absorbance a t 261 mp after the induction period-e.g., the latter portions of the curves of Figure 3-can be obtained from plots according to Equation 5. The evaluated apparent first order rate constants for the appearance of the chromophores a t 225 mp and 261 mp for various temperatures with lo-3.V 2-deoxyribose in 0.244.M HC1 are O C . , 105ky~s,105kza: 60.0°, 2.60, 0.662; 70.0°, 9.72, 2.16; 75.0", 17.6, 2.80: 80.0°, 31.2, 5.68 where the k values are in seconds-'. The apparent heat of activation, A H , for the rate of appearance of the 225 mp absorbance is 27,600 cal/mole. It is 23,300 cal/mole for the 261 mp absorbance. From this information and a rate constant at a specific temperature
r
0.26 HRS.
0.9
0.8
0.7
0.6 Y
2
0.5
0.4
03
02
01
0.0 260
280 M O WAVELENOTH ( M P I
Y O
Figure 5. Example of effect and 1.7 X 1 O - W N a O H a t ultraviolet spectrum of mixture 10-4M 2-deoxyribose and 1 OP5Muracil
of time 2 3 " on
of 5 X 5 X
bimolecular rate constants, k A + = k/aH + can be estimated for various temperatures in accordance with
where R = 1.987 cal degree-' mole-' and T = 273 " C. The log P for k H + for the 225 mp chromophore is 14.24 and is 10.61 for the 261 nip chromophore. At low temperatures, viz. 50" C., and low acid concentrations, viz. O,OlL$' HC1, only the 225 n i p chromophore appears obtainable. Thus, a lower limit of conditions is imposed. An upper limit is imposed by the fact that different products than that product which is responsible for the 261 mp chromophore appear a t high acidities (6, '7, 1 2 , 1 2 ) and the boiling point temperature is technologically infeasible. The conditions of 80" C. and IN HC1 are suitable on these bases and from the point of view of minimal time of assay. The final absorbance a t 261 mp is reached a t 5 hours and maintains constancy for a t least 24 hours. The 261 is directly promp absorbance, .I,, portional to deoxyribose concentration [ U ] , as is clearly shown in Figure 2. The apparent molar absorptivity of degraded deosyribose under these conditions is c = .-1/[D] = 1300 where statistical analysis of the regression of .I on [ D ] gives a atandard deviation of the molar absorlltivity ue = 0.001 and the standard deviation about regression is U A = 0.012. 'Thus the error in the direct assay of a pure sample of deosyribose is ca. 1%. Concentrations as low
+
as 10-4M can be assayed where initial concentrations higher than 10-3.11 should be diluted before applying the analytical procedure and the calibration curve of Figure 2. Properties of Degraded DeoxyOF ribose. . \ L K A L I N E DZSTRUCTIOK DE G R A D E D 2- n E O X Y R I B 0 S E c H R O M 0P H O R E . T h e chromophore of degraded deoxyribose obtained by t h e described methods is stable under the acidic condition-: cited. When the solution is made alkaline, the 261 mp chromophore (.t in Figure 1) shifts to 293 mp ( B in Figure 1). This shift is irreversible since on re-acidification, the 261 mp absorbance is lost (C in Figure 1). The 293 mp absorbance in alkaline solution disappears as shown (D in Figure 1) by a first order process. The complete loss of the alksline-effecled degraded-deosyribose chromophore with timr in the presence of The uracil is shown in Figure 5 . apparent first order rate constants calculated in accordance with Equation 5 are directly proportional to the hydroxyl ion concentration (Figure 6) so that the bimolecular rate constants kOH- = k/ [ S a O H ] can be calculated. The apparent first order rate constants for the destruction of the 293 mp chromophore of acid-degraded 2-deoxyribose (5 X 10-4.V) in 8.5 X 10-4-Tf S a O H are: "C., 104k; 27.7, 1.88; 29.2, 1.97; 34.5, 2.67; 37.5, 3.01; 45.0, 4.77 where the k values are in second-'. The heat of activation for the destruction of the degraded deoxyribose chromophore in accordance with Equation 6 is A H , = 9,650 cal/mole and the log P value for koH- is 4.37. The apparent rate constants a t a given [NaOH] were invariant with degraded deosyribose concentration. CHLOROFORM E X T R A C T I O N O F 261 mp CHROMOPHORE OF DEGRADED DEOXYRIBOSE. The acid generated chromophore obtained by the methods cited is extractable with chloroform and may be spectrophotometrically assayed a t 261 mp against a chloroform blank. The 261 my absorbance, A, is directly proportional to deosyribose concentration [D],as is clearly shown in Figure 2. The apparent molar absorptivity (for a total volume of CHCI, twice that of aqueous acid estracted) is e = .4 / [D] = 516 where statistical analysis of the regression of .-I on [ D ] gives a standard deviation of the molar absorptivity u I = 0.002 and the standard deviation about regression U A = 0.028. .is in the direct assay, concentrations as low as 10-4M can be assayed whereas initial concentrations higher than 10-3.V should be diluted before applying the analytical procedure. ASSAYOF DEOXYRIBOSE I N PRF:SE:NCE OF C H n o ~ i o P H o n E s . 2-Deosyribose can be readily assayed in the presence of
Figure 6. Linear dependence of apparent first order rate constants, k in seconds-' for disappearance of 293 mp chromophore of acid-degraded 7 X 10-4M 2-deoxyribose as function of [NaOH] a t 27.7"
chromophores that are stable under the acid and alkaline conditions derived for the assay. X mixture of deoxyribose and uracil, even to the extent of 1 0 : l mixture, can be assayed for both components by spectrophotometry a t 261 mp using the acid degradation procedure. The subsequent spectrophotometric assay at 293 mp for uracil (after alkaline degradation of the absorbance contribution of acid-degraded deoxyribose as in Figure 5 ) permits the assay of deoxyribose by the "difference technique" given in the Esperimental. The series of mixtures of 2-deosyribose and uridine which were prepared for analysis are given in Table I. Cridine is stable under the given assay conditions ( 4 ) . The results of the assay of duplicates are also given. The variation among dulllicate assays was negligible. The mean value of all the deoxyribose assays for the direct spectrophotometric method was 95.8y0 of the prepared composition with a standard deviation of for the direct spectrophotometric assay. The mean value of all the deoxyribose assays for Table I. Analysis of 2-Deoxyribose in Deoxyribose-Uridine Mixtures
Found 104[D]
Composition ___- CHCI, lo4 104 ex[Urilo4 [Uritrac- dine] [D] diiie] Ihrect tion Direct 2 00 2 00 2 00 4 00 6 00 8 00
6 4 2 2 2 2
95 85 99
00 00 00 00
1 1 1 3
00 00
5 55 7 70
90
2 10 2 15 2 05 4 13 6 24 8 20
VOL. 37, NO. 2, FEBRUARY 1965
6 4 2 2 2 2
03
14
00 00 02 03
273
the chloroform extraction method was 104.0y0 of the prepared composition with a standard deviation of 2.2% for a single assay. The series of mixtures of 2-deoxyribose and deoxyuridine which were prepared for direct spectrophotometric assay are given in Table 11. Deosyuridine is not completely stable under the given assay conditions (4). When the deoxyuridine :deoxyribose ratio is low (I., Advances i n Carbohydrate Chemistry 8 , 45 (1953), Academic Press, New York.
Table II. Direct Spectrophotometric Analysis of 2-Deoxyribose in Deoxyribose-Deoxyuridine Mixtures
Composition lo4 [DeoxyFound, 10‘ (D] uridine] la4 [D] 8.00 2.00 8.07 6.08 6.00 2.00 2.00 3.99 4.00 2.00 2.17 2.00 4.00 2.W 2.00 2.00 6 00 2.18” a These are corrected values for solvolysis of deoxyuridine during assay on premise that observed uracil is equimolar with generated deoxyribose. If this correction is not made, 2.78 X 104M deoxyribose is the assay value.
(11) Pacsu, E., Hiller, L. A., J . A m . Chem. Soc. 70,523 (1948). (12)Rice, F. A , . Fishbein,, L... Zbid.. 78. ’ 1005 (1956). ’ (13) Webb, J. A l . , Levy, H. B., J . Biol. Chem. 213, 107 (1955). I
,
RECEIVEDfor review October 5, 1964. Accepted December 2, 1964. This investigation was supported in part by an institutional grant t o the University of Florida by the American Cancer Society and in part by Grant GM-09864-02,03 from the Kational Institutes of Health, U. S. Public Health Service, Bethesda, hld.
Determination of Free Urea in Ethyl Stearate Adduct by X-Ray Diffraction D. A. LUTZ, J. J. HUNTER, and C. R. EDDY Eastern Utilization Research and Development Division, Pa. 19118
b A method for the direct determination of free urea in urea adducts of ethyl stearate by x-ray diffraction was developed. Known amounts of the urea and urea-free ethyl stearate adducts were mixed as dry powders and pressed into disks. These disks were reground and repressed until random orientation was obtained. The ratio of the height of the 4.0 A. peak of free-urea to that of the 4.1 A. peak of the adduct was calculated by statistical methods for each mixture in the series. A graph of these ratios plotted against the per cent free urea was used as a standard curve for the determination of free urea in unknowns, Ethyl stearate was chosen only as a representative guest molecule for this study. In principle, this method should be applicable to any type of inclusion compound when host and adduct are crystalline solids. 274
ANALYTICAL CHEMISTRY
S
U. S.
Department o f Agriculture, 600 East Mermaid Lane, Philadelphia,
INCE THE DISCOVERY
of urea adducts
(1) in 1940 by Bengen, they have
become the subject of much serious work. One of the chief difficulties in quantitative studies of the physical properties of these adducts is the presence of free urea in unknown amounts. Since the crystal structure of urea is different from that of its adducts (5), x-ray diffraction (3) can be used for estimating the amount of free urea. Herrmann (9) has used x-ray analysis for assaying the amount of adduct from the intensity of adduct diffraction maxima, with reference to a specimen presumed to be pure. This would be insufficiently sensitive to determine small quantities of free urea that would interfere with studies of physical properties, since urea would have to be determined by difference. We have developed a method for direct determination of free urea from
the ratio of heights of a free urea maximum and a n adduct maximum. I n order to develop the details of the procedure, known amounts of urea and urea-free ethyl stearage adducts were mixed as dry powders and pressed into disks for x-ray examination. EXPERIMENTAL
Preparation of Adduct. Following method 2 of Swern ( 7 ) 1 gram of ester was added to a solution of 5 grams of urea in 20 ml. of absolute methanol and 3 ml. of isopropanol. The mixture was heated until clear. The solution was allowed to cool to room temperature and the precipitate filtered off. The precipitate was washed three times with absolute methanol to dissolve any excess urea and three times with benzene to dissolve any excess ester. X-ray diffraction indicated that this washing procedure was adequate since lO-fold
