THE JOURNAL cm BIOLC~ICAL CHEMISTRY

Vol. 244, No. 6, Issue of March 25, PD. 160%1607,1969

Printed in U.S.A.

The Effect of Deuterium Oxide on the Fluorescence of

Reduced Nicotinamide Adenine Dinucleotide Free in
Solution and in Complexes with Liver
Alcohol Dehydrogenase*
(Received for publication, September 19, 1963)

CHARLES H. BLOMQUIST
From the Biomedical Research Group, Los Alamos Scientific Laboratory, University of California, Los Alamos,
New Mexico 87544

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SUMMARY for the study of dehydrogenase-coenzyme complexes. Changes
in coenzyme absorption and fluorescence emission with the forma-
The fluorescence emission intensity at 460 nm of reduced
nicotinamide adenine dinucleotide in solution is increased tion of binary and ternary enzyme and enzyme-inhibitor com-
in the presence of deuterium oxide over that observed in plexes are well known for liver alcohol dehydrogenase (9).
water. The increase in fluorescence intensity is a linear In this report data are presented showing the effect of DzO
on the fluorescence emission of NADH, reduced 3-acetylpyridine
function of DzO concentration, and in 75 volume % DzO
equals 20%. A similar increase is observed with N-methyl- adenine dinucleotide, N-methyldihydronicotinamide, and phos-
dihydronicotinamide, reduced 3-acetylpyridine adenine phodiesterase-hydrolyzed NADH. The effects of complex
dinucleotide, and phosphodiesterase-hydrolyzed NADH. formation with liver alcohol dehydrogenase on the sensitivity of
The DzO perturbation of NADH fluorescence is reduced in NADH to DzO are also shown.
binary complexes of NADH-alcohol dehydrogenase and in
MATERIALS AND METHODS
ternary complexes of NADH-alcohol dehydrogenase-in-
hibitor. Fluorescence measurements were made on an Aminco-Bowman
The data indicate that the altered sensitivity of the bound spectrophotofluorometer fitted with a constant temperature
coenzyme to DzO results from an interaction of the nico- cuvette holder. All data were obtained with cuvettes, 10 x 10
tinamide moiety with the enzyme protein and is not simply mm, and solution temperatures were measured with a YSI
the result of unfolding of the coenzyme molecule with com- 42SL Tele-Thermometer (Yellow Springs Instrument Company,
plex formation. Inc., Yellow Springs, Ohio). A 150-watt xenon arc (Engelhard-
Hanovia, Inc., Newark, New Jersey) was used as the exciting
light source. Entrance and exit slits were adjusted to allow a
negligible sca.ttered light contribution to the spectra. Spectra
were recorded manually and corrected for blank fluorescence.
A 5- to 20-~1 aliquot of NADH solution was added to a sample
The technique of fluorescence perturbation spectroscopy (l), containing buffer, additives, and Hz0 or DzO.
which is similar in principle to solvent perturbation difference Horse liver alcohol dehydrogenase (twice crystallized sus-
spectroscopy (2), has been used to estimate the relative solvent pension, Worthington) was routinely treated as previously
exposure of tyrosine and tryptophan residues in a number of described (3). One sample was purified by chromatography on
proteins. Changes in the solvent perturbation of chromophore carboxymethyl cellulose (10). Enzyme concentration, expressed
fluorescence can be indicative of conformational changes (1, 3). as micronormal in active centers (pN = 2 X PM), was determined
The effects of substrates or inhibitors on the solvent fluorescence by titration with NAD in the presence of excess pyrazole (11).
perturbation of aromatic amino acids in lysozyme (4, 5) and Isobutyramide and n-butyramide were products of Distillation
Staphylococcus uureus nuclease have been reported (6). Solvent Products Industries (Rochester, New York). NAD+ and
perturbation techniques have also been applied to chromophoric NADH were obtained from Calbiochem. Reduced 3-acetyl-
residues such as dyes (7) and inhibitors (8) which can be intro- pyridine adenine dinucleotide was obtained from P-L Biochem-
duced into proteins. This approach would appear to be of value icals. The purity of NAD+ and NADH was estimated by reduc-
* This work was performed under the auspices of the United tion with ethanol and liver alcohol dehydrogenase at pH 10.0 or
States Atomic Energy Commission. oxidation with propionaldehyde and liver alcohol dehydrogenase

1605
1606 Deuterium Oxide Effect on CoenxymeFluorescence Vol. 244, No. 6

at pH 7.0. Concentration determinations were based on molar TABLE I

absorbances of 18.0 X 1Oa M? cm-l at 260 nm for NADf, 6.22 X Effect of D20 on fluorescenceof NADH, related compounds,
lOa M-I cm-r at 340 nm for NADH, and 9.1 X lOa M-l cm-l at and enzyme-bound NADH
363 nm for 3-acetylpyridine NADH.
Compound COllditiOllS
N-Methyldihydronicotinamide was prepared by the method .-
of Karrer and Blumer (12) from N-methylnicotinamide chloride &Da0
(K and K Laboratories). A molar absorptivity of 7.0 X 1Oa NADH Phosphate, pH 7.0, 75 1.197 f 0.018 (6)
~-1 cm-l at 360 nm (13) was used to determine concentration. /A = 0.1
For phosphodiesterase hydrolysis of NADH, an extract of 3-AP-NADH* Phosphate, pH 7.0, 75 1.225 f 0.022 (2)
lyophilized Crotah &ma&us venom (Rose Allen Reptile p = 0.1
Institute, Silver Springs, Florida) was used. A l-ml reaction N-Methyldihy- 0.1 M Tris, pH 9.0 75 1.242 f 0.019 (7)
dronicotina-
mixture containing 0.9 pmole of NADH, 2 pmoles of MgC12,
mide
and 0.2 mg of venom extract in phosphate buffer, p = 0.1, pH
NADH-ADH” Phosphate, 0.04 M, 75 1.062 f 0.029 (5)
7.0, was incubated at 37” for 30 min. An aliquot of this reac- pH 7.4
tion mixture was taken for fluorescence measurements. NADH-ADH- Phosphate, 0.04 M, 75 1.005 f 0.015 (4)
Reagent grade salts and glass-distilled water were used. isobutyramide pH 7.4, or G =
Deuterium oxide (l&O, 99.8%) was distilled under vacuum 0.1, pH 7.0
before use. NADH-ADH-n- Phosphate, pH 7 .O, 65 0.996 f 0.010 (2)
butyramide p = 0.1
RESULTS -
a Average value f standard deviation; number of determina-

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Fluorescence emission spectra of NADH in Hz0 and DzO are tions indicated in parentheses.
presented in Fig. 1A. The principal effect of DzO (75 volume %) * 3-AP-NADH, 3-acetylpyridine NADH.
c ADH, alcohol dehydrogenase.

is an increase in fluorescence intensity. The ratio of emission

intensity in DzO to that in Hz0 at the wave length of maximum
,. . .
Hz0 emission is written as Fmo:Frrzo. For NADH, in the con-
centration range of 2 to 10 pN, the value of FDa,:FBfi at 460 nm
was 1.197 =t 0.018 (average of six determinations, &standard
deviation). No shifts in excitation or emission msxima were
observed in DzO.
To test the possibility that DzO enhancement of fluorescence
was due to a shift in the equilibrium distribution of the NADH
between open and folded conformations, solvent effects on the
fluorescence of phosphodiesterase-hydrolyzed NADH and N-
methyldihydronicotinamide were examined. N-Methyldihydro-
L nicotinamide was excited at 360 nm with the fluorescence emis-
sion maximum observed at 460 nm. As shown in Table I, the
fluorescence intensity at the emission maximum for both com-
pounds increased in DzO to the same extent as observed with
NADH. The completeness of the phosphodiesterase hydrolysis
under the conditions of the experiment was established by the
absence of a 260 nm maximum in the fluorescence excitation
spectrum.
The DQO enhancement of the fluorescence of a number of
chromophores containing proton donor groups has been ex-
plained on the basis of a kinetic isotope effect on proton transfer
reactions in the excited state (14). Such a mechanism involving
the amino group of the carboxamide side chain of NADH could
account for the DsO effect. As seen in Table I, the fluorescence
of 3-acetylpyridine NADH (excitation at 360 nm, emission maxi-
01’
360 ’ ’
400 ’ ’
440 ’ ’
400 ’ ’
520 ’ ’
560 ’ ’
600 ’I- mum at 480 nm) is enhanced to the same extent as NADH.
WAVELENGTH (nm) With the formation of enzyme-coenzyme and enzyme-coen-
zyme-inhibitor complexes, the fluorescence enhancement ob-
FIG. 1. Effect of Da0 on the fluorescence emission and excita-
tion of NADH and liver alcohol dehydrogenase-NADH-isobutyra- served in DzO is markedly reduced (Fig. 1B and Table I). In
mide. A, emission spectra, excitation at 340 nm, 8.1 pN NADH the experiments with the binary complexes, 80 to 85% of the
in4mMphosphate,pH7.4,24.5”; B,9.8p~ LADH,S.~~NNADH, coenzyme was bound and with the ternary complexes 95 %. For
14.9 ells isobutyramide in 4 mM phosphate buffer, pH 7.4, 24.5”, the complexes, fluorescence was excited at 330 nm with emission
excitation at 330 nm; 0, Hz.0; l , 75 volume To DlO; inset, de-
pendence of Fn,o:Fn%o on volume ‘% DzO; 9.8 pi NADH in phos- recorded at 430 nm. It can be seen that the major reduction in
phate buffer, pH 7.0, p = 0.1, 22”. NADH sensitivitv to DzO occurs with the formation of the binary
Issue of March 25, 1969 C. H. Blomquist 1607

complex. Formation of ternary complexes with fatty acid amide solvent sensitivity of the bound coenzyme with ternary complex
inhibitors, which are competitive with aldehyde substrates (9), formation are at the limit of resolution of the method. Clearly,
results in complete loss of the DzO response. no conclusions can be drawn from these data regarding the struc-
ture of the binary and ternary complexes until the mechanism of
DISCUSSION the D20 perturbation of NADH is known.
The data presented show that the fluorescence intensity at 460
Acknowledgments-The author is grateful to Mr. D. L. Wil-
nm of the dihydronicotinamide moiety of NADH is increased liams for the vacuum distillation of the deuterium oxide and to
approximately 20% in 75 volume y0 D20 over that observed in Drs. C. T. Gregg, P. M. Kraemer, and D. G. Ott for helpful die-
HzO. An equivalent increase is observed with N-methyldihy-
cussions.
dronicotinamide and phosphodiesterase-hydrolyzed NADH, in-
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