Journal o f Radioanalytical Chemistry VoL 3 (1969) 3 - - 9

GENERAL PRINCIPLES OF ENZYMATIC METHODS AND THEIR USE IN THE DETERMINATION

OF THE RADIOCHEMICAL PURITY OF MONO- AND DISACCHARIDES LABELLED WITH 14C

G. I. MEEROV

V. M. Bechterev Psychoneurological Institute, Leningrad ( U S S R )

(Received March 29, 1968, revised October 11, 1968)

General principles of enzymatic methods for the determinat ion of radiochemi- cal purity of natural ~4C-mono- and -disaccharides are suggested, and analytical schemes using these principles have been worked out. The enzymatic method of deter* mining the radiochemical purity of a4C-glucose is given as an illustration.

Various kinds of chromatography and electrophores have been used so far as the conventional methods for the determination of radiochemical purity of 14C-mo- nosaccharide and l~C-disaccharide preparations. In the present paper enzymatic methods are suggested for checking the radiochemical purity of 14C-mono- and -disaccharides.

Principle of enzymatic methods for the radiochemical purity analysis of natural ~4C-monosaccharides

The suggested principle is based on the quantitative enzymatic phosphorylation of l~C-monosaccharides (Table 1) by means of a suitable specific phosphokinase enzyme. In the reaction phosphate esters of 14C-monosaccharides are formed which are precipitated from the solution as barium salts ;* the remaining radioactivity of the solution depends on the radiochemical admixtures** which have been present as impurities.

By comparing the radioactivity of the preparation before and after the action of the enzyme and precipitation of the phosphate esters, we can establish the exact percentage of radioactivity ascribable to the main ~C-compound and that due to different radioadmixtures.

Table 1 lists the 14C-monosaccharides (in stated combinations, the ~4C labeling may be at any position in the molecule) that can be analysed for radiochemical purity with the use of this principle; the suitable enzymes are also shown.

* The quantitat ive removal of monosaccharide phosphate esters as practically insoluble bar ium salts is widely used in biochemical analysis 1-4.

** In this paper we use the term 'radiochemical admixtures ' to denote various organic 1~C-compounds which are present in 14C-monosaccharide and 14C-disaccharide preparations.

1" J. Radioanal. Chem. 3 (1969)

4 G. I, M E E R O V : G E N E R A L P R I N C I P L E S OF E N Z Y M A T I C M E T H O D S

Table 1 is based on the da ta o f DIXON and WEBB) All enzymes listed in Table 1 bu t mannok inase were avai lable in high pur i ty t - lZ ; hexokinase was even recrystal- l ized 5'6's (in Table 1 it is m a r k e d by an asterisk).

The enzymat ic me thod worked out by the au thor for the de te rmina t ion o f rad io- chemical pur i ty o f 14C-glucose by means o f hexokinase serves as an exper imenta l i l lus t ra t ion of the suggested pr inciple .

Table 1

Scheme for the determination of radiochemical purity of natural 14C-monosaccharides

No. Radioact ive substrate

analysed

D-1~C-Glucose

D-UC-Fructose

D-x~C-Mannose

D-a4C-Galactose D-~aC-Ribose D-Arabinose

Specific enzyme

Glucokinase

Fructokinase (Ketohexokinase)

Mannokinase

Galactokinase Ribokinase Arabinokinase

N o t e s

If the preparation is known to contain neither l~C-fructose nor 14C-mannose, or when these have been determined in the preparation by some other method, hexokinase may be used

If the preparation is known to contain neither xaC-glucose nor 14C-mannose, or when these have been determined in the preparation by some other method, hexokinase may be used

If the preparation is known to contain neither x~C-glucose nor ~4C-fructose, or when these have been determined by some other method, hexokinase may be used

* We used a crystalline hexokinase preparation obtained from yeast (Lewson Co., England).

Enzymatic method for the determination of the radiochemical purity of 14C-glucose preparations

A 0.010 M so lu t ion o f ~4C-glucose (0.2 ml) with a to ta l act ivi ty o f 0 . 3 5 - 0 . 7 0 #Ci, a 0.025 M solu t ion (0.2 ml) o f pre l iminar i ly neut ra l ized adenos ine t r iphospha te (ATP) , 0.025 M MgCI 2 (0.2 ml), 0.2 M bora te buffer o f p H 7.3 (0.2 ml) and hexo- k inase (0.2 ml)* (1 mg o f h e x o k i n a s e dissolved in 0.2 ml o f 0.2 M bora te buffer o f p H 7.3) were p laced into a 10-ml centr ifuge tube. The tube was kep t in a water

J. Radioanal. Chem. 3 (1969)

G. I. MEEROV: GENERAL PRINCIPLES OF ENZYMATIC METHODS 5

thermostate at 37 ~ C for 1 hr. Then 1.5 ml of 5 % ZnSO4* and 1.5 ml of 0.3 N Ba(OH)2 were added to the mixture and, after 20 min, the sample was centrifuged (3000 rpm) for 15 rain. The supernatant was then separated, and 0.1 ml of it was applied onto a planchet of 16 mm diameter. The supernatant from one sample was applied to 3 parallel planchets that were dried in a thermostate at 75 ~ C for 2 hrs. Two parallel test samples were used for the analyses. The standard samples were prepared similarly to the test ones, however, without incubation with the enzyme. Determination of the radioactivity of the standard required 3 min, and that of the test samples 10 rain. An end-window counter T-25 (window thickness 1 mg/cm 2) connected to a radiometer type B-2 was used. The percentage radioactivity due to radiochemical admixtures in the a~C-glucose preparation (Q) can be calculated by the following formula *~'

Asol

Q = Atot ng I ~ k V Ato t ! j

where:

Ato t -

Aim p -

Ag], sol -

Aso I

mgl, sed - -

C

Z

ng I -

F/g1, s e d -

_Rg 1

K -

V -

Total activity of the preparation found in the sample (cpm) (Ato t =

= Ag I -~- Aimp). Activity of radioadmixtures in the preparation (cpm). Activity of laC-glucose (or its phosphate Ba-salt) in the solution af ter precipitation of the greater part of 14C-glucose-6-phosphate Ba-salt (cpm) (Agl, sol = k V " Rgl). Activity of the solution (cpm) (A~ol = Agl, ~,1 + Aimp)"

Activity of the precipitated 14C-glucose-6-phosphate Ba-salt (cpm)-

(Agl, sed = Atot - - As,;l). Amount of 14C-glucose (M)taken for the analysis (weight). In our exper- iment, c = 2 .10 -8 M Contents of the basic substance in the preparation (part of the whole). In the analysed preparation Z = 0.951. Amount of a4C-glucose (M) taken for the analysis (rig 1 = c" Z ) . In our experiment n~I = 1.902 �9 10 -6 M. Quantity of the precipitated ~4C-glucose-6-phosphate Ba-salt (M). Specific activity of glucose (cpm/M). Value of saturation (solubility) of ~4C-glucose-6-phosphate Ba-salt. (In our case it was equal to 1.07.10 -5 M)***

Volume of solution (1). In our experiment, V = 0.C04 1.

* The ZnSOa and Ba(OH)2 solut ions mus t be adequate ly p repa red ~3. ** The au thor is deeply grateful to Professoc T. SCHONFELD, Universi ty of Vienna, for his

cr i t ical remarks and help in working out the formula. *** The value o f K was es t imated by using two chemical ly and radiochemical ly pure I~C-

glucose preparat ions .

3". Radioanal. Chem. 3 (1969)

6 G. I. M E E R O V : G E N E R A L P R I N C I P L E S O F E N Z Y M A T I C M E T H O D S

The original formula is

Q = Aim p" 100 _ (Aso I - Agl, sol) " I 0 0 = (Aso I - kV" Rgl) " 100 (1)

Atot Atot Atot

Based on the specific activities, the a4C-glucose-6-phosphate precipitated in our experiment was equal to that of l~C-glucose. Thus, if the value of ng 1 is known, Rg I c an be derived from the following formula:

Agl sed Atoi -- Asol Rg 1 = ~ ' �9 - ( 2 )

/'/gl, sed ng 1 -- kY Substituting Rgl into Eq. (1) and performing some rearrangements, we obtain Eq. (3) which is convenient for further calculations.

Q = Atot ngl-kV ~ �9 100 (3)*

In the experiment performed, ng I >~ kV. The results of the radiochemical purity determination of a commercial 1-14C -

glucose preparation are presented in Table 2. The data obviously show the applicability of the suggested principle to determine

the radiochemical purity of 14C-glucose.

Table 2

Dete rmina t ion of radiochemical purity of a 14C-glucose commercial prepara t ion

1~1o.

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15

Radioact iv i ty o f s tandard

sample , c p m

2595 2617 2617 2617 2617 2617 2349 2349 2349 2349 2413 2413 2413 2413 2413

Radioact iv i ty o f test sample,

c p m

93 97 94 79 85 87 89 77 70 83 82 79 88 81 83

Percentage o f radio-

admixtures

1.38 1.49 1.38 0.77 0.97 1.08 1.59 1.08 0.67 0.28 1.08 0.97 1.38 1.18 1.18

Ari thmet ic m e a n

1.16

Quadra t ic m e a n

• 0.259

M e a n error

-k- 0.067

Note: The mean error of radioactivi ty de terminat ion 1~ for s tandard samples is about q - l . l - - l . 3 ~ and for test samples •

* The author considers tha t the formula in its general form can also be used for determin- ing the radioactivity percentage due to radioadmixtures in preparat ions of other 14C-mono- saccharides analysed for the determinat ion of radiochemical purity using enzymatic methods.

J. Radioanal. Chem. 3 (1969)

G . I . M E E R O V : G E N E R A L P R I N C I P L E S O F E N Z Y M A T I C M E T H O D S 7

0 r

t.~ " 0 o ~

t~

0

.m "o

~ . ~ o ~ . ' ' ~

0 .,~ ~ 0 ~ ~ . �9 ,~,~..~

~ 0 o ,~ . ~ ' ~ o o ~ ~ '

O ' n ~ ~ ~ O , - - ~ n:~ ~ ' " , .~ ~ ~

0 0 ~ ~ n , . O 0 e~ ~ I~ ~ ~ ~ 0

~ - ~ " =oN o==~' . . - , - ~ , . = n ~ , o - z -

, - o

" o

O

02

~ , ~ ._~ .~ ,~ o ..~

o ~ . ~ . . . ~ . . o o

o r/)

~ .2 o

c c ~ o

J. RadioanaL Chem. 3 (1969)

8 G.I . MEEROV: GENERAL PRINCIPLES OF ENZYMATIC METHODS

General principle of enzymatic methods for radiochemical purity analyses of natural 14C-disaecharides

The suggested principle is based on the total hydrolysis of the l~C-disaccharide to be analyzed by means of a suitable glycosidase. As a result of this reaction, 14C-monosaccharides are formed which undergo total quantitative enzymatic phos- phorylation with the corresponding phosphokinase enzyme; the phosphate esters of the monosaccharides are then removed from the medium as barium salts. The radioactivity remaining in the solution will be due to the radiochemical admixtures.

The principle is suitable for the radiochemical purity analyses of l~C-disaccha- rides, independently from the place of ~4C-labelling.

Table 3 presents the scheme of radiochemical purity determination for the five most important natural ~4C-disaccharides. Table 3 is based on the data of the mono- graph of DIXON and WEBB ~.

Each enzyme included in the table was available in high purity ~5-2~ Table 3 shows that hexokinase is a very effective agent for most of the ~4C-disac-

charides, owing to its capability of phosphorylating D-glucose, D-fructose and D-mannose 5.

Discussion

For the determination of radiochemical purity of 14C-glucose we used hexoki- nase as the phosphorylating enzyme. Hexokinase can phosphorylate not only D- glucose, but also D-fructose and D-mannose 5. Glucokinase would be a more spe- cific enzyme for this purpose, for it phosphorylases D-glucose selectively 9, but for our experiments no highly purified glucokinase preparation was available. The explorer having highly purified glucokinase preparation at hand can use this meth- od to obtain absolute specificity. The suggested enzymatic method for the deter- mination of radiochemical purity of 14C-glucose, the scheme of Table 1 and the methods for obtaining highly purified preparations of several phosphokinases 6-12 make the usage of the general principle quite perspective for working out simple and reliable enzymatic methods of the determination of radiochemical purity of other ~4C-monosaccharides. The same method and the scheme of analyses given in Table 3 along with worked out methods of making highly purified glycosidase preparations ~5-2~ make it basically possible to work out simple and reliable methods for the determination of radiochemical purity of ~4C-disaccharides.

References

I. R. K. CRANE, A. SoLs, Animal tissue hexokinase, in S. P. COLOWICK and N. O. KAPLAN, Methods in E n z y m o l o g y , Vol. 1, p. 277, Academic Press, New York, 1955.

2. H. G. HERS, Fructokinase (Ketohexokinase), in S. P. COLOWICK, N. O. KAPLAN, Me thods in Enzymology, Vol. 1, p. 266, Academic Press, New York, 1955.

3. L. F. LELOIR, R. N. TRUCCO, Galactokinase and galactowaldenase, in S. P. COLOWICK and N. O. KAPLAN, Methods in Enzymology, Vol. 1, p. 290, Academic Press, New York, 1955.

4. H. Z. SABLE, Yeast pentokinase, in S. P. COLOWICK and N. O. KAPLAN, Methods in Enzy- mology, Vol. 1, p. 357, Academic Press, New York, 1955.

J. Radioanal. Chem. 3 (1969)

G. I. MEEROV: GENERAL PRINCIPLES OF ENZYMATIC METHODS 9

5. M. DIXON, E. WEBB, Enzymes, London, Longmans, 1958. 6. M. ROHDEWALD, Phosphatiibertragende Enzyme (Transphosphatasen), in HOPPEE SEYLER/

THIERFELDER, Handbuch der physiologisch- und pathologisch-chemischen Analyse, Berlin Bd. VI/b, p. 528, 1966.

7. R. E. PARKS, E. BEN-GERSHAM, H. A. LARDY, J. Biol. Chem., 227 (1957) 231. 8. R. A. DARROW, S. P. COLOWICK, Hexokinase from baker's yeast, in S. P. COLOWICK and

N. O. KAPLAN, Methods in Enzymology, Academic Press, New York--London, Vol. V, p. 226, 1962.

9. R. L. ANDERSON, M. Y. KAMEL, Glucokinase II. Aerobacter aerogenes, in S. P. COLOWICK and N. O. KAPLAN, Methods in Enzymology, Vol. X, p. 388, Academic Press, New York, 1966.

10. M. R. HEII'~RICH, S. HOWARD, Galactokinase, in S. P. COLOWlCK and N. O. KAPLAN, Methods in Enzymology, Vol. IX, p. 407, Academic Press, New York, 1966.

l l .B . W. AGRANOff, A. GINSBURG, Ribo-deoxyribokinases, in S. P. COLOWICK and N. O. KAPLAN, Methods in Enzymology, Vol. V, p. 302, Academic Press, New York, 1962.

12. W. A. VOLK, D-Arabinokinase, in S. P. COLOWICK and N. O. KAPLAN, Methods in Enzy- mology, Vol. IX, p. 442, Academic Press, New York, 1966.

13. S. P. COLOWICK, N. O. KAPLAN, Methods in Enzymology, Vol. III, p. 85, Academic Press, New York, 1957.

14. I. I. IVANOV, V. K. MOGHISTOV, YU. M. SHTUKKEIBERG, E. F. ROMANCZEV, E. I. VOROBJEV. Radioactive Isotopes in Medicine and Biology, Moscow, 1955. (In Russian.)

15. K. MYRB~CK, Invertases, in The Enzymes (edited by P. O. BOYER, H. LARDY and K. MYRB.KCK) 2nd ed., Vol. IV, p. 379, Academic Press, New York, 1960.

16. K. WALLENEELS, H. DIEKMANN, Glykosidasen, in HOPPE-SEYLER/THIERFELDER, Handbuch der physiologisch und pathologisch-chemischen Analyse, Bd. VI/b, p. 1156, Berlin, 1966.

17. J. LARNER, Other Glucosidases, in The Enzymes (edited by P. O. BOYER, H. LARDY and K. MYRB.~CK) 2rid ed., Vol. 4, p. 369, Academic Press, New York, 1960.

18. J. E. COURTO~S, F. PETER, a-Galactosidase from coffee beans, in S. P. COLOWICK and N. O. KAPLAN, Methods in Enzymology, Vol. VIII, p. 565, Academic Press, New York, 1966.

19. H. HALVORSON, c~-Glucosidase from yeast, in S. P. COLOWlCK and N. O. KAPLAN, Methods in Enzymology, Vol. VIII, p. 559, Academic Press, New York, 1966.

20. K. WALLENFELS, fl-Galactosidase, in S. P. COLOWICK and N. O. KAPLAN, Methods in Enzymology, Vol. V, p. 212, Academic Press, New York, 1962.

J. Radioanal. Chem. 3 (1969)