NAD+-pyridoxine and NAD+-pyridoxamine complexes

258

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26498 NAD+-PYRIDOXlNE

AND NAD+-PYRIDOXAMINE

COMPLEXES

V. D. ANAND AND W. R. CARPER Gamey Research Center, Department of Chemistry, Wichita State University, Wichita, Kam 672o8 (U.S.A .) (Received October 5th, 197o)

SUMMARY I. N A D + - p y r i d o x i n e a n d N A D + - p y r i d o x a m i n e complexes were s t u d i e d b y fluorescence t e c h n i q u e s at 3 °° over a p H range of 8.4-IO.O. 2. The polarization factor was seen to increase with increasing N A D + concent r a t i o n in all cases. 3. Fluorescence quenching studies e s t a b l i s h e d large complex f o r m a t i o n cons t a n t s for b o t h complexes. 4. T h e N A D + - p y r i d o x i n e complexes are e n d o t h e r m i c in n a t u r e while the NAD~'p y r i d o x a m i n e complexes are s t r o n g l y e x o t h e r m i c .

D u r i n g a kinetic s t u d y of t h e e n z y m e glucose d e h y d r o g e n a s e 1 it was n o t e d t h a t p y r i d o x i n e a n d p y r i d o x a m i n e b o t h a c t e d as c o m p e t i t i v e inhibitors. As both of these molecules h a v e p r o n o u n c e d fluorescence p r o p e r t i e s it a p p e a r e d t h a t binding studies b e t w e e n these c o m p o u n d s a n d t h e various c o m p o n e n t s of the e n z y m a t i c assay m i g h t be possible. Such a s t u d y was e n c o u r a g e d b y t h e success of fluorescence as an a n a l y t i c a l tool 2-~ a n d b y the use of quenching studies as an i n d i c a t i o n of complex formation1, 5-11. PARKER AND REES 11 p o i n t e d out t h a t if one uses dilute solutions such t h a t the a m o u n t of a b s o r b e d light is small, t h e n Eqn. I relates fluorescence i n t e n s i t y to concentration : F -- ~I0(2.3o 3 ecd)~ [¢11

(I)

E x p e r i e n c e has shown 3 t h a t at c o n c e n t r a t i o n s less t h a n 1.10 -8 M, Eqn. I is a p p r o p r i a t e , a n d t h a t c o n s e q u e n t l y t h e r e is a linear r e l a t i o n s h i p between the log of c o n c e n t r a t i o n a n d t h e log of fluorescence intensity. Here caution m u s t be exercised, as Eel m a y well v a r y due to i n t e r n a l r e a b s o r p t i o n , etc. 2-4. However, assuming t h a t tile v a r i a t i o n in t h e log t e r m r e m a i n s trivial, it is t h e n possible to j u s t i f y the linear log fluorescence vs. log c o n c e n t r a t i o n plots t h a t follow. Such a linear relationship t h e n m a k e s it possible to establish s t a n d a r d plots which can be used to d e t e r m i n e complex f o r m a t i o n c o n s t a n t s w h e r e v e r either fluorescence quenching or enhancem e n t occurs. Polarized fluorescence can also be used to establish b i n d i n g b e t w e e n two or m o r e molecules p r o v i d i n g t h a t their c o m b i n e d m o l e c u l a r dimensions are such t h a t t h e i r t u m b l i n g r a t e r e l a t i v e to t h e i r fluorescence lifetime in solution is not e x t r e m e l y Biochim. Biophys. Acta, 230 (I971) 258--26I

N A D + - P Y R I D O X I N E AND N A D + - P Y R I D O X A M I N E

259

rapid1,6, 9. The possible existence of this effect involves the rate of fluorescence decay and consequently the lifetime of the excited state. If a slow moving complex is excited with plane polarized light, the resulting fluorescence will be polarized with its electric vector perpendicular to the activating beam. Conversely, small molecules in low viscosity solvents assume random orientations during their excited state lifetimes and thus their fluorescence is largely depolarized. The degree of molecular association can then be related to the polarization factor (p)12 which increases as complexation occurs: FBE P

-

-

FI] + F L

£.E

- -

(2)

FEE + FEn FBB

where FII ( = FEE) is the fluorescence intensity measured with the polarizing prisms orientated with electric vectors parallel. F± ( = FEB) is the intensity measured with the prisms crossed. The ratio (FBE/FBB) is a correction factor which accounts for the selective transmission of the emission monochromator (B polarized). If this ratio were not used, the observed spectrum would be shifted approximately 2 nm to longer wavelengths 13. Chemicals. A grade NAD ~, pyridoxine and pyridoxamine were purchased from Calbiochem. In order to remove NADH from NAD +, the NAD+ was eluted from a DEAE-cellulose column and recrystallized according to the method of WINER14. The resulting NAD+ showed no fluorescing NADH and was considered to be pure. Triply distilled water was passed through appropriate columns to insure removal of ions and organic matter. The instrmnent used was sentitive to less than io parts per billion of quinine sulfate dissolved in o.o 5 M H2SO 4 and no observable fluorescence was detected in any of the water used throughout this investigation. All measurements were taken oil an Aminco-Bowman spectrophotofluorometer equipped with an ellipsoidal mirror system and Glan-Thompson prisms. Only freshly prepared solutions were used and o.o 5 M sodium barbital was used as the buffer. The experiments were repeated several times to insure consistency. Bulk solutions of I . l O -5 M pyridoxine or pyridoxamine were diluted to I . I O -~ M by the addition of varying amounts of buffer and NAD +. The range of NAD+ covered from I-IO -a to 5" lO-3 M in lO% increments. TABLE I FLUORESCENCE SPECTRA (nm) AT 3 o°.

pH

~max(excitation) Amax(emission)

t~ridoxine 8.4 9.2 IO.O

332 320 315

4o5 396 390

Pyridoxamine 8.4 9.2 IO.O

331 320 315

406 398 385

Biochim. Biophys. Acta, 230 (1971) 258-261

260

v.D.

A N A N D , W. R. C A R P E R

The spectral characteristiscs of both pyridoxine and pyridoxamine are given in Table I. These were the wavelength maxima observed and used during the course of this investigation. As might be expected, both give similar spectra in terms of both band energies and fluorescence intensity. TABLE

li

F L U O R E S C E N C E Q U E N C H I N G AND POLARIZATION OF N A D + - - P Y R I D O X I N E

24AD + conch. (M)

Pyridoxine fluorescence

Polarization factor

0-5" 10--2 0. 5 • 10 -a 0.5" 10 .4 0.5" 10 -5 O.O

39"4 51.3 60.0 63'2 66.0

0"0439 O.O414 0.0392 0"0345 O.O313

TABLE

( 5 . O ' I O -~ M )

SO L U T I O N S

Ili

F L U O R E S C E N C E Q U E N C H I N G AND POLARIZATION OF N A D + - - P Y R I D O X A i M I N E ( 5 . O . 1 0 - 6 l~t) SO L U T I O N S

NAD+

conch. (M)

Pyridoxamine Polarization fluorescence factor

0. 5 • I o -2 0 . 5 ' IO--3 0 . 5 • IO -4

0. 5 " I o o.o

TABLE

5

39.6 54.7

0.0477 0.0450

58,0

0.0430

62.1 64.0

0.0420 o.o416

IV

F O R MAT IO N CONSTANTS AND E N T H A L P I E S OF T H E N A D + - P Y R I D O X I N E

pH

Temp.

K

8. 4 8.4 9.2 9.2 io.o IO.O

3°o 4°° 3°0 4°° 32° 4°°

48.7 83-4 116 276

TABLE

COMPLEX

A H (kcal/mole) 2~ -4~ 4-

6.9 5 .2 4.2 6.5 7 9 7 4 - 28 1329 4- 57

io.i 16. 3 12.2

V

F O RMA T ION CONSTANTS AND E N T H A L P I E S OF T H E N A D + - - P Y R I D O X A M I N E COMPLEX

pH

Temp.

8.4 8.4 9.2 9.2 lO.O io.o

3 °0 4°0 3 °0 4 °0 3°0 4°o

K

AH(kcal/mole) --

147 47.5 444 165

4444--

Biochim. Biophys. Acta,

-9 3.9 I2 i1

--21.3 --18.8

23 ° ( I 9 7 I) 2 5 8 - 2 6 1

N A D + ± P Y R I D O X I N E AND N A D + - P Y R I D O X A M I N E

261

A J o b ' s analysis indicated a m a x i m u m fluorescence quenching effect at I : I mole ratios in all cases. Consequently all of the following data have been analyzed accordingly. It should also be pointed out that analyses based upon other ratios give formation constants that are not independent of concentration. Sample polarization and fluorescence data are given in Tables If and I I I . These results were obtained at 3 °0 in solutions of p H 9.2. The increase in the polarization factor coupled with the simultaneous quenching of fluorescence present strong arguments for complex formation. Tables IV and V contain the formation constants that were determined from the quenching of pyridoxine or pyridoxamine fluorescence. The NAD+-pyridoxine complex decreases in strength with decreasing pH, however its endothermic enthalpy is greatest in the vicinity of pH 9.2. This suggests an ionization process on the part of either NAD +, pyridoxine, or possibly both. A final point concerns the fluorescence of pyridoxamine in a pH 8. 4 or 8.6 solution with NAD +. At both p H ' s the fluorescence is quite complex as it increases to a certain point during the titration process and then starts to decrease. A number of experiments were run using different concentration combinations, and the initial results were verified. The fluorescence inflection point is in the vicinity where the ratio of NAD + to pyridoxamine is greater than 8ooo to I. At this point fluorescence quenching begins and continues until the end of the titration process. These results are consistent with a small formation constant which one may obtain by extrapolating from the results at other p H ' s given in Table V. In view of past evidence l-a, it appears that a complicated competition process is occurring and that this process m a y involve Na +, barbital ions or perhaps even different forms of NAD + (refs. 15, 16). ACKNOWLEDGMENTS

The authors wish to thank Professor Robert K. Gholson of the Biochemistry Department, Oklahoma State University for helpful discussions and the generous donation of an initial crystalline sample of pure NAD+. Financial support from the University Research Fund is also acknowledged. REFERENCES I M. R. PAULE, A. 123 (I968) 8.

J.

ANDREOLI, M. A. CARPER AND M. R. CARPER, Arch. Biochem. Biophys.,

2 S. UI~DENFRIEND,FluorescenceAssay in Biology and Medicine, Academic Press, New York, 1962. 3 D. M. HERCULES, Fluorescence and Phosphorescence Analysis, Interscience, New York, 1966. 4 B. L. VAN DUIJREN, Chem. Rev., 63 (1963) 325 . 5 S. F. VELICK, in W. D. MCELROY AND B. GLASS, Light and Life, The J o h n s H o p k i n s Press, 6 7 8 9 Io II 12 13 14 15 16

Baltimore, 1961. S. F. VELICK, J. Biol. Chem., 233 (1958) 1455. G. D. SHORT AND C. A. PARKER, Spectrochim. Acta, 23A (1967) 2487. Izi. KNIBBE, K. ROLLIG, F. P. SCHAFER AND A. J. WELLER, J. Chem. Phys., 47 (1967) 1184M. A. CARPER AND W. R. CARPER, J. Chem. Ed., 45 (1968) 662. g. S. SOLOMON, C. STEEL AND A. VVTELLER,Chem. Commun., (1969) 927 . C. A. PARKER AND W. T. REES, Analyst, 85 (196o) 587 . A. C. ALBRECHT, J. Mol. Speetry., 6 (1961) 84. T. AZUMI AND S. P. McGLYNN, J. Chem. Phys., 37 (1962) 2413. A. D. WINER, J. Biol. Chem., 239 (1964) 3598. O. JARDETZKY AND iNT. G. WADE-JARDETZKY, J. Biol. Chem., 241 (1966) 85. D. G. CRoss AND 171. F. FISHER, Biochemistry, 8 (1969) 1147.

Biochim. Biophys. Acta, 230 (I971) 258 261