- Email: [email protected]
[3] Temperature dependence of the spectroscopic properties of NADH
8
NICOTINIC ACID" ANALOGS AND COENZYMES
[3]
vettes. The fluorescence is measured at an emission wavelength of 430 nm, with excitation at 370 nm. Calculations ng/ml I - M e t h y l n i c o t i n a m i d e fluorescence of sample - fluorescenc e of bl a nk - f l u o r e s c e n c e of standard - fluorescence of s t a nda rd bl a nk x 100 ng/ml ng/ml N i c o t i n a m i d e fluorescence of sample - fluorescenc e of bl a nk = fluorescence of standard - fluorescence of standard bl a nk x 70.8 ng/ml
Comments Since NAD + and NADP + also yield fluorescent products in this procedure, it is necessary that nonhemolyzed serum samples be utilized for measurement of 1-methylnicotinamide and nicotinamide. In the quantitative conversion of nicotinamide to 1-methylnicotinamide using iodomethane, the reaction must occur under anhydrous conditions to prevent formation of HI which converts nicotinamide to the unreactive pyridinium hydroiodide derivative. The extraction of nicotinamide from a neutral, NaCl-saturated aqueous solution into ethyl acetate, after a prior clean-up extraction with ethyl acetate at pH - 1 (nicotinamide is present as the nonextractable hydrochloride in 0.5 N HC1), ensures residue-free extracts for the iodomethane reaction.
[3] T e m p e r a t u r e
Dependence
of the Spectroscopic
Properties of N A D H By
ALAN D. B. MALCOLM
Introduction The ultraviolet (UV) absorption, fluorescence on excitation at 260 nm, nuclear magnetic resonance, and circular dichroic spectra of the reduced nicotinamide coenzymes all show greater temperature dependence than expected. This is attributed to an equilibrium between a closed and an open conformation of NAD(P)H where higher temperatures favor the open conformation, a An understanding of this conformational change 1 S. F. Velick, in " L i g h t and L i f e " (W. D. M c E l r o y an d B. Glass, eds.), p. 108. J ohns H o p k i n s Press, Baltimore, Maryland, 1961.
METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3
[3]
TEMPERATURE EFFECTS ON N A D H SPECTRA
9
and its effect on the spectroscopic properties is important for three reasons. The UV absorption and fluorescence of NAD(P)H are widely used for the assay of many enzymes--not only dehydrogenases, but also many other enzymes that may be coupled to a suitable dehydrogenase. Attempts to discover the structure of NAD(P)H in aqueous solution must clearly allow for this, and finally, it seems as though there may be a correlation between the conformation of the coenzyme when bound to the enzyme and whether the enzyme is an A or B side dehydrogenase. Ultraviolet Spectrum The UV spectrum of NAD(P)H shows two maxima--one at 260 nm contributed by the adenine ring and the other close to 340 nm which arises from the reduced nicotinamide ring. The original determination z of the extinction coefficient gave 6.18 ± 0. ll × 10a M -1 cm -1 but the temperature was not specified. Although the extinction coefficient at 366 nm is only about 50% of that at 340 nm, the higher wavelength is often used tbr spectrophotometric measurements because of the existence of an intemse line in the mercury discharge lamps used as early UV sources. Similarly, 334 nm was also used. A suggestion 3 that a modification of atomic absorption spectroscopy using spectral lines of rhodium (343.5 nm) or chromium (357.8 nm) might be used for the determination of NAD(P)H does not appear to have been adopted and, therefore, no details of these wavelengths will be discussed. Hohorst 4 first pointed out that tlhe extinction coefficient at 366 nm is temperature dependent, whereas that at 340 nm is not. A more detailed study 5 showed that whereas the percentage variation in extinction coefficient at 340 nm is not large enough to be worth making corrections, the change at 366 nm is significant. The table summarizes some of the most relevant data on this. z,4-r It should be noted that the final line r of this table contains the recommended temperature and wavelengths of the International Federation of Clinical Chemistry (I.F.C.C.). The variations in extinction coefficients have been used to calculate 2 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). E. J. Harris,FEBS Lett. 4, 160 (1969). 4 H. J. Hohorst, Biochem. Z. 328, 509 (1956). 5 A. D. B. Malcolm, Anal. Biochem. 55,278 (1973). n H.-U. Bergmeyer, ed., "Methoden der enzymatischen analyse," 1st ed., p. 27. Vedag Chemie, Weinheim, 1962. r H.-U. Bergmeyer, G. N. Bowers, M. Horder, and D. W. Moss, Clin. Chim. Acta 70, FI9 (1976).
10
NICOTINIC ACID" ANALOGS AND COENZYMES
[3]
EXTINCTION COEFFICIENTS OF N A D H AT VARIOUS WAVELENGTHS AND TEMPERATURES
E × 103(M-lcm 1) Temp. (°C)
334 nm
339 nm
340 nm
366 nm
Not given 25 1 25 15 25 35 30
---6.0 ---6.18
-------6.3
6.18 _+ 0.11 6.29 6.29 -6.23 6.16 6.07 --
-3.30 3.36 3.30 3.53 3.30 3.07 --
References a
a Numbers refer to text footnotes.
a n e n t h a l p y c h a n g e for the c l o s e d to o p e n c o n f o r m a t i o n of b e t w e e n +33 a n d +63 kJ m o l - L 5
Fluorescence, Circular Dichroism, and Nuclear Magnetic Resonance N A D ( P ) H f l u o r e s c e s at a r o u n d 470 n m o n e x c i t a t i o n at e i t h e r 260 n m or 340 n m . W h e n it is the a d e n i n e m o i e t y that is excited, f l u o r e s c e n c e o c c u r s o n l y after r a d i a t i o n l e s s e n e r g y t r a n s f e r to the n i c o t i n a m i d e group. 1 This e n e r g y t r a n s f e r has a v e r y m u c h higher p r o b a b i l i t y in the " f o l d e d " or " c l o s e d " c o n f o r m a t i o n t h a n in the " o p e n " o n e , a n d this e x p l a i n s the g r e a t e r t e m p e r a t u r e d e p e n d e n c e of f l u o r e s c e n c e e x c i t e d at 260 n m comp a r e d with that arising a f t e r 340 n m i l l u m i n a t i o n . W h e n f l u o r e s c e n c e is u s e d for the a s s a y o f n i c o t i n a m i d e - d e p e n d e n t e n z y m e s the o p t i m a l w a v e l e n g t h d e p e n d s o n the w a v e l e n g t h v a r i a t i o n of the r a d i a t i o n e m i t t e d b y t h e lamp. H o w e v e r , s i n c e f l u o r e s c e n c e c a n o n l y b e u s e d q u a n t i t a t i v e l y in c o n j u n c t i o n with a s t a n d a r d , t e m p e r a t u r e a n d w a v e l e n g t h v a r i a t i o n s are n o t u s u a l l y a p r o b l e m . U s i n g a t e m p e r a t u r e j u m p a p p a r a t u s with f l u o r e s c e n c e d e t e c t i o n , it has b e e n s u g g e s t e d that the c h a n g e from " c l o s e d " to " o p e n " o c c u r s in a b o u t 1 m s e c . 8 It has n o t p r o v e d p o s s i b l e , h o w e v e r , to r e p e a t t h e s e o b s e r v a t i o n s . 5 F l u o r e s c e n c e m e a s u r e m e n t s give a n e n t h a l p y c h a n g e for the c o n f o r m a t i o n a l c h a n g e of a b o u t 10 kJ m o l - L 5 T h e c h e m i c a l shifts o f the p r o t o n s in the a d e n i n e a n d n i c o t i n a m i d e a G. Czerlinski and F. Hommes, Biochirn. Biophys. Acta 79, 46 (1964).
[4]
PURIFICATIONOF COMMERCIALNADH
11
rings also vary with temperature, and a value of +23 kJ mo1-1 for the separation of the two rings has been calculated. 9 The circular dichroism of NADH also varies with temperature, TM and by using data obtained at 260 nm the enthalpy for the closed to open change has been shown to be 69 kJ mo1-1) These variations in enthalpy suggest that a simple two-conformation model may be an oversimplification, that NAD(P)H can best be described as ~, multiplicity of complexes undergoing rapid exchange, and that different spectroscopic techniques may be observing different equilibria. 5,9 O. Jardetzky and N. E. W. Jardetzky, J. Biol. Chem. 241, 85 (1966). 10 D. W. Miles and D. W. Urry, J. Biol. Chem. 243, 4181 (1968).
[4] P u r i f i c a t i o n o f C o m m e r c i a l
NADH
By WOLFGANGLOESCHE, I. WENZ, U. TILL, H. PETERMANN, and A. HORN Accurate measurements of the activities of enzymes requiring NADH as a coenzyme largely depend on the purity of the NADH. Therefore, many authors have been engaged in problems of NADH purity, the stability of NADH preparations, and the formation of inhibitors. Reviews are given by Gerhard et al.1 and others, z From these data it is obvious that difficulties are encountered in purifying NADH and assuring its stability. Chromatographic purification procedures commonly used do not completely remove the inhibitors2 "4 Furthermore, removal of the eluent from NADH after chromatographic purification often leads to NADH degradation with the formation of inhibitory compounds. The stability of the purified product is unfavorably influenced by humidity. In previous papers we described a chromatographic procedure using DEAE-Sephadex with potassium bicarbonate (KHCO3) as an eluent s'6 W. Gerhardt, B. Kofoed, L. Westlund, and B. Parlu, Scand. J. Clin. Lab. Invest., Suppl. 33,, 139 (1974). z G. Anido, S. B. Rosalki, E. J. van Kampen, and M. Rubin, eds., "Quality Control in Clinical Chemistry." de Gruyter, Berlin, 1975. a S. A. Margolis, B. F. Howell, and R. Schaffer, Clin. Chem. 23, 1581 (1977). 4 E. Haid, P. Lehmann, and J. Ziegenhorn, Clin. Chem. 21,884 (1975). s A. Horn, U. Till, W. Loesche, and W. Achilles, GDR Patent WP B 01 d/170 506 (1973). 6 W. Loesche, R. Bublitz, A. Horn, W. Koehler, H. Petermann, and U. Till, J. Chromatogr. 92, 166 (1974).
METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3
NICOTINIC ACID" ANALOGS AND COENZYMES
[3]
vettes. The fluorescence is measured at an emission wavelength of 430 nm, with excitation at 370 nm. Calculations ng/ml I - M e t h y l n i c o t i n a m i d e fluorescence of sample - fluorescenc e of bl a nk - f l u o r e s c e n c e of standard - fluorescence of s t a nda rd bl a nk x 100 ng/ml ng/ml N i c o t i n a m i d e fluorescence of sample - fluorescenc e of bl a nk = fluorescence of standard - fluorescence of standard bl a nk x 70.8 ng/ml
Comments Since NAD + and NADP + also yield fluorescent products in this procedure, it is necessary that nonhemolyzed serum samples be utilized for measurement of 1-methylnicotinamide and nicotinamide. In the quantitative conversion of nicotinamide to 1-methylnicotinamide using iodomethane, the reaction must occur under anhydrous conditions to prevent formation of HI which converts nicotinamide to the unreactive pyridinium hydroiodide derivative. The extraction of nicotinamide from a neutral, NaCl-saturated aqueous solution into ethyl acetate, after a prior clean-up extraction with ethyl acetate at pH - 1 (nicotinamide is present as the nonextractable hydrochloride in 0.5 N HC1), ensures residue-free extracts for the iodomethane reaction.
[3] T e m p e r a t u r e
Dependence
of the Spectroscopic
Properties of N A D H By
ALAN D. B. MALCOLM
Introduction The ultraviolet (UV) absorption, fluorescence on excitation at 260 nm, nuclear magnetic resonance, and circular dichroic spectra of the reduced nicotinamide coenzymes all show greater temperature dependence than expected. This is attributed to an equilibrium between a closed and an open conformation of NAD(P)H where higher temperatures favor the open conformation, a An understanding of this conformational change 1 S. F. Velick, in " L i g h t and L i f e " (W. D. M c E l r o y an d B. Glass, eds.), p. 108. J ohns H o p k i n s Press, Baltimore, Maryland, 1961.
METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3
[3]
TEMPERATURE EFFECTS ON N A D H SPECTRA
9
and its effect on the spectroscopic properties is important for three reasons. The UV absorption and fluorescence of NAD(P)H are widely used for the assay of many enzymes--not only dehydrogenases, but also many other enzymes that may be coupled to a suitable dehydrogenase. Attempts to discover the structure of NAD(P)H in aqueous solution must clearly allow for this, and finally, it seems as though there may be a correlation between the conformation of the coenzyme when bound to the enzyme and whether the enzyme is an A or B side dehydrogenase. Ultraviolet Spectrum The UV spectrum of NAD(P)H shows two maxima--one at 260 nm contributed by the adenine ring and the other close to 340 nm which arises from the reduced nicotinamide ring. The original determination z of the extinction coefficient gave 6.18 ± 0. ll × 10a M -1 cm -1 but the temperature was not specified. Although the extinction coefficient at 366 nm is only about 50% of that at 340 nm, the higher wavelength is often used tbr spectrophotometric measurements because of the existence of an intemse line in the mercury discharge lamps used as early UV sources. Similarly, 334 nm was also used. A suggestion 3 that a modification of atomic absorption spectroscopy using spectral lines of rhodium (343.5 nm) or chromium (357.8 nm) might be used for the determination of NAD(P)H does not appear to have been adopted and, therefore, no details of these wavelengths will be discussed. Hohorst 4 first pointed out that tlhe extinction coefficient at 366 nm is temperature dependent, whereas that at 340 nm is not. A more detailed study 5 showed that whereas the percentage variation in extinction coefficient at 340 nm is not large enough to be worth making corrections, the change at 366 nm is significant. The table summarizes some of the most relevant data on this. z,4-r It should be noted that the final line r of this table contains the recommended temperature and wavelengths of the International Federation of Clinical Chemistry (I.F.C.C.). The variations in extinction coefficients have been used to calculate 2 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). E. J. Harris,FEBS Lett. 4, 160 (1969). 4 H. J. Hohorst, Biochem. Z. 328, 509 (1956). 5 A. D. B. Malcolm, Anal. Biochem. 55,278 (1973). n H.-U. Bergmeyer, ed., "Methoden der enzymatischen analyse," 1st ed., p. 27. Vedag Chemie, Weinheim, 1962. r H.-U. Bergmeyer, G. N. Bowers, M. Horder, and D. W. Moss, Clin. Chim. Acta 70, FI9 (1976).
10
NICOTINIC ACID" ANALOGS AND COENZYMES
[3]
EXTINCTION COEFFICIENTS OF N A D H AT VARIOUS WAVELENGTHS AND TEMPERATURES
E × 103(M-lcm 1) Temp. (°C)
334 nm
339 nm
340 nm
366 nm
Not given 25 1 25 15 25 35 30
---6.0 ---6.18
-------6.3
6.18 _+ 0.11 6.29 6.29 -6.23 6.16 6.07 --
-3.30 3.36 3.30 3.53 3.30 3.07 --
References a
a Numbers refer to text footnotes.
a n e n t h a l p y c h a n g e for the c l o s e d to o p e n c o n f o r m a t i o n of b e t w e e n +33 a n d +63 kJ m o l - L 5
Fluorescence, Circular Dichroism, and Nuclear Magnetic Resonance N A D ( P ) H f l u o r e s c e s at a r o u n d 470 n m o n e x c i t a t i o n at e i t h e r 260 n m or 340 n m . W h e n it is the a d e n i n e m o i e t y that is excited, f l u o r e s c e n c e o c c u r s o n l y after r a d i a t i o n l e s s e n e r g y t r a n s f e r to the n i c o t i n a m i d e group. 1 This e n e r g y t r a n s f e r has a v e r y m u c h higher p r o b a b i l i t y in the " f o l d e d " or " c l o s e d " c o n f o r m a t i o n t h a n in the " o p e n " o n e , a n d this e x p l a i n s the g r e a t e r t e m p e r a t u r e d e p e n d e n c e of f l u o r e s c e n c e e x c i t e d at 260 n m comp a r e d with that arising a f t e r 340 n m i l l u m i n a t i o n . W h e n f l u o r e s c e n c e is u s e d for the a s s a y o f n i c o t i n a m i d e - d e p e n d e n t e n z y m e s the o p t i m a l w a v e l e n g t h d e p e n d s o n the w a v e l e n g t h v a r i a t i o n of the r a d i a t i o n e m i t t e d b y t h e lamp. H o w e v e r , s i n c e f l u o r e s c e n c e c a n o n l y b e u s e d q u a n t i t a t i v e l y in c o n j u n c t i o n with a s t a n d a r d , t e m p e r a t u r e a n d w a v e l e n g t h v a r i a t i o n s are n o t u s u a l l y a p r o b l e m . U s i n g a t e m p e r a t u r e j u m p a p p a r a t u s with f l u o r e s c e n c e d e t e c t i o n , it has b e e n s u g g e s t e d that the c h a n g e from " c l o s e d " to " o p e n " o c c u r s in a b o u t 1 m s e c . 8 It has n o t p r o v e d p o s s i b l e , h o w e v e r , to r e p e a t t h e s e o b s e r v a t i o n s . 5 F l u o r e s c e n c e m e a s u r e m e n t s give a n e n t h a l p y c h a n g e for the c o n f o r m a t i o n a l c h a n g e of a b o u t 10 kJ m o l - L 5 T h e c h e m i c a l shifts o f the p r o t o n s in the a d e n i n e a n d n i c o t i n a m i d e a G. Czerlinski and F. Hommes, Biochirn. Biophys. Acta 79, 46 (1964).
[4]
PURIFICATIONOF COMMERCIALNADH
11
rings also vary with temperature, and a value of +23 kJ mo1-1 for the separation of the two rings has been calculated. 9 The circular dichroism of NADH also varies with temperature, TM and by using data obtained at 260 nm the enthalpy for the closed to open change has been shown to be 69 kJ mo1-1) These variations in enthalpy suggest that a simple two-conformation model may be an oversimplification, that NAD(P)H can best be described as ~, multiplicity of complexes undergoing rapid exchange, and that different spectroscopic techniques may be observing different equilibria. 5,9 O. Jardetzky and N. E. W. Jardetzky, J. Biol. Chem. 241, 85 (1966). 10 D. W. Miles and D. W. Urry, J. Biol. Chem. 243, 4181 (1968).
[4] P u r i f i c a t i o n o f C o m m e r c i a l
NADH
By WOLFGANGLOESCHE, I. WENZ, U. TILL, H. PETERMANN, and A. HORN Accurate measurements of the activities of enzymes requiring NADH as a coenzyme largely depend on the purity of the NADH. Therefore, many authors have been engaged in problems of NADH purity, the stability of NADH preparations, and the formation of inhibitors. Reviews are given by Gerhard et al.1 and others, z From these data it is obvious that difficulties are encountered in purifying NADH and assuring its stability. Chromatographic purification procedures commonly used do not completely remove the inhibitors2 "4 Furthermore, removal of the eluent from NADH after chromatographic purification often leads to NADH degradation with the formation of inhibitory compounds. The stability of the purified product is unfavorably influenced by humidity. In previous papers we described a chromatographic procedure using DEAE-Sephadex with potassium bicarbonate (KHCO3) as an eluent s'6 W. Gerhardt, B. Kofoed, L. Westlund, and B. Parlu, Scand. J. Clin. Lab. Invest., Suppl. 33,, 139 (1974). z G. Anido, S. B. Rosalki, E. J. van Kampen, and M. Rubin, eds., "Quality Control in Clinical Chemistry." de Gruyter, Berlin, 1975. a S. A. Margolis, B. F. Howell, and R. Schaffer, Clin. Chem. 23, 1581 (1977). 4 E. Haid, P. Lehmann, and J. Ziegenhorn, Clin. Chem. 21,884 (1975). s A. Horn, U. Till, W. Loesche, and W. Achilles, GDR Patent WP B 01 d/170 506 (1973). 6 W. Loesche, R. Bublitz, A. Horn, W. Koehler, H. Petermann, and U. Till, J. Chromatogr. 92, 166 (1974).
METHODS IN ENZYMOLOGY, VOL. 66
Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3