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Spectrophotometric analysis of rat liver lactate dehydrogenase-coenzyme and coenzyme analog complexes
ARCHIVES OF BIOCHEMISTBY
AND BIOPHYSICS
127, 566-575 (196%
Spectrophotometric
Analysis
Dehydrogenase-Coenzyme Analog
of Biochemistry,
and Coenzyme
Complexes
CARL S. VESTLING Department
of Rat Liver Lactate
AND
University
1
ULRICH of Iowa,
Iowa
KUNSCH City, Iowa 52240
Received March 22, 1968; accepted May 11, 1968 A study of difference spectra in the range, 300-500 rnr is reported involving highly purified rat liver lactate dehydrogenase (LDH) and oxidized or reduced coenzyme or coenzyme analog (nicotinamide-adenine dinucleotide (NAD) or acetylpyridine-adenine dinucleotide ( AcPy AD). The formation of the binary complexes, LDH . NAD or LDH AcPyAD is associated with the development of spectral bands with A,,, at 335 rnr and 355 rnp, respectively. The observed absorbance is proportional to coenzyme concentration at fixed LDH concentration. The spectral behavior of the LDH .oxidized coenzyme binary complexes is clearly distinguishable from that of the binary complexes involving NADH or AcPyADH. A conclusion is that the binding of oxidized coenzymes to LDH results in a significant change in the chromophoric behavior of a hybridized form of the substituted pyridine ring. The formation of abortive ternary complexes involving LDH . oxidized coenzyme pyruvate or LDH oxidized coenzyme . a-ketobutyrate was accompanied by slow progressive spectral changes which reached equilibrium values, dependent on the concentration of all three reactants. For LDH NAD pyruvate A- was 323 mr; for LDH AcPyAD pyruvate A, was 341 mp. The spectral changes were fully reversible upon dialysis vs. AcPyAD-buffer in the one case but not upon dialysis vs. NAD-buffer in the other. Complete recovery of enzyme activity was noted in each case after dialysis to remove pyruvate or ol-ketobutyrate. The spectral changes are considered to be those associated with the allcsteric binding of pyruvate or a-ketobutyrate to binary complexes of LDH . oxidized coenzyme with re sulting conformational changes in LDH which affect the active sites. No abortive ternary complex involving LDH-reduced coenzyme (or reduced coenzyme analog) -lactate could be detected by spectral means.
Spectral studies from various laboratories have produced reports of enzyme-reduced coenzyme (or reduced coenzyme analog) complexes (6, 9, 15, 19, 22, 25, 26). These studies involved the measurement of difference spectra in the case of several dehydrogenases and indicated relatively small spectral shifts when free reduced coenzyme was compared to bound coenzyme (spectral reduced
range: 300400 my). The interpretation of such experiments has generally been that the act of binding of reduced coenzyme exerted a relatively minor influence on the chromophoric behavior of the dihydropyridine residue of the molecule. On the other hand there has appeared only one preliminary report, which the present authors have noted, of spectral studies of the effect of binding of oxidized coenzyme (or analog) to a dehydrogenase (28). In this case the author described a very small spectral change when bovine
’ This work was supported by Grant CA 07617 from the National Cancer Institute, National Institutes of Health. 568
LACTATE
DEHYDROGENASE-COENZYME
heart lactate dehydrogenase (LDH)’ was combined with NAD, but a very marked change when the enzyme was combined with the acetyl pyridine analog of NAD (AcPyAD). Winer (28) reported a X,,, for the binary complex, LDH . AcPyAD, of 355 rnp at pH 8.15. The experiments which are described by the present authors serve to confirm and extend Winer’s observations and lend themselves to the interpretation that the act of binding of AcPyAD or NAD to LDH is one in which there is a decided influence of the enzyme on the chromophoric system of the oxidized coenzyme or analog (which in the free state show very little absorbance in the spectral region, 300400 mp). Fromm and co-workers (10, 11, 19) were the first to call attention to “abortive” ternary complexes formed from enzyme, coenzyme and “wrong substrate” (ex., LDH . NAD . pyruvate). Winer (28) reported a similar result and indicated that the X, for the abortive ternary complex formed from bovine heart LDH, NAD, and pyruvate was 325 rnp under the same conditions mentioned above. Recently Gutfreund et al. (12) reported the formation of pig heart LDH . NAD + pyruvate complexes in a study of the inhibition of LDH by pyruvate. They measured increases in absorbance at 325 rnr and correlated spectral changes with fluorescence quenching and inhibition. The observations of Gutfreund et al. lend support to the results by the present authors. Anderson (1) performed some preliminary experiments involving rat liver LDH, NAD or AcPyAD and pyruvate and laid the foundation for the present study. Anderson, Florini and Vestling (2) interpreted their kinetic measurements on the basis of abortive ternary complexation of LDH . NAD . pyruvate but could demonstrate no such effect ‘Abbreviations: LDH, Rat liver lactate dehydrogenase; NAD, NADH, Nicotinamide adenine dioxidized nucleotide, and reduced; AcPyAD, AcPyADH, Acetylpyridine adenine dinucleotide, oxidized and reduced. A, Absorbance.
569
COMPLEXES
with the components: LDH . NADH . lactate. In the present studies a series of spectral determinations is described and a plausible interpretation proposed. In addition, an unusual time dependence has been found for spectral changes accompanying the formation of abortive ternary complexes. These spectral changes are fully reversible in the case of AcPyAD, but not in the case of NAD. No loss of enzyme activity is encountered after dialysis to remove the “wrong substrate” in either case. A time dependence has also been noted for spectral changes accompanying binary complex formation when NADH but not AcPyADH is involved. EXPERIMENTAL
PROCEDURES
RAT LIVER LACTATE DEHYDROGENASE Rat liver LDH was isolated, purified and assayed according to Hsieh and Vestling (14). The enzyme used in these experiments had a specific activity of 100 f 4 I.U./mg of protein, which value corresponds to that for maximally pure enzyme. This LDH is a tetramer of the M, type (21) with a molecular weight of 126,000 (8). Before each experiment 1.6 ml of LDH solution was dialyzed at 0’ under N2 against 120 ml of a 0.02 M potassium phosphate buffer, pH 7.8, which was 0.02 M with respect to ammonium sulfate and 10.” M with respect to 2-mercaptoethanol. Five changes of buffer were made during a 4-hour period. This buffer was used for all spectrophotometric determinations. At the end of the dialysis aliquots were taken out and assayed. The protein concentration was calculated from the absorbance at 280 rnr = 12.6) and from the measurement of en(E:%, zymatic activity [l mg of pure LDH is equivalent to 101.5 International Units (rmoles NADH ml-’ mini’].
COENZYMES The following materials were analyzed according to Ciotti and Kaplan (7). 1. NAD: /%NAD. 3.5 HzO, 987, pure, Sigma Chemical Company, found to be 97-98:;’ pure. 2. AcPyAD: AcPyAD ‘2 HZO, 91”; pure, P.L. Biochemicals, Inc., found to be 90% pure. 3. NADH: fl-Na?NADH .3 H20, 98% pure, Sigma Chemical Company, found to be 91% pure.
570
VESTLING
-----Sam Bea --uI II
AND KijNSCH
4. AcPyADH: BaAcPyAD .4 Hz0 prepared in this laboratory by reduction of 91% pure AcPyAD . 2 Hz0 (P.L. Biochemicals, Inc.) with yeast ADH, and found to be 83% pure. 5. NasAcPyADH. 2Hz0, 93% pure (P.L. Biochemicals, Inc.), was found to be 86% pure.
II
SUBSTRATES 1. Lactate: Lithium D, L-lactate, anhydr., was prepared in this laboratory from 85% pure D, Llactic acid (17) and found to be 97% pure in a potentiometric titration in an organic solvent mixture (18). 2. Pyruvate: Na-pyruvate, 99% pure, Sigma Chemical Company.
--
Reference
Beam
1
SCHEME
OTHER COMPOUNDS P-Mercaptoethanol (Eastman) was redistilled in uacuo (bp 57-58”, 14 mm). The resulting vg was 1.5011 as compared to the value of 1.4996 (5). It was stored under nitrogen in l-ml ampoules. Dialysis tubing: Visking, lo-mm diameter, flat. Sections were placed in boiling distilled water during two 30-minute periods, to remove ultraviolet absorbing material. All other materials used were of analytical reagent grade.
WAVELENGTH
(mu)
FIG. 1. Difference spectra. Curve A, LDH (3 x 10m6M) vs. buffer. Curve B, AcPyAD (6 X 1O-5 M) vs. buffer. Curve C. sodium pyruvate (10-j M) vs. buffer.
SPECTROPHOTOMETRIC PROCEDURE A double tandem system of matched cuvettes was used in a Cary Model 15 recording spectrophotometer (cuvette volume, 1.5 ml; light path, 1.0 cm). The cuvette arrangement is sketched in Scheme 1. The cuvette numbers will appear in the legends of the appropriate figures. The cuvette positioning and alignment were carefully checked. All spectral measurements were made at 25 f 1’. Constant temperature was maintained by circulation of water through the jacketed cuvette compartment of the spectrophotometer. The cuvettes used were closed with discs cut from gum rubber “serum caps” attached with gum rubber sleeves. Measured volumes of appropriate solutions were added through the caps from calibrated syringes. The final volume in each cuvette was 1.0 ml for all binary complex systems. Additions of 10 ~1 were made to form the ternary complexes; the resulting volume changes . were within l?; and were neglected. RESULTS
The data to be presented are grouped in four sections. Complexation involving AcPyAD precedes that of NAD, and that involving AcPyADH precedes that of
A
X,OX.
WAVELENGTH
33Bmu
(mu)
FIG. 2. Binary and ternary systems involving AcPyAD. Difference spectra, double tandem system, pH 7.8, 25”. Curve A. A,, 355 mp. Sample beam: I. LDH(3 X 10-"M), ACP~AD(~ X lo-"M).II. Buffer. Reference beam: III. LDH (3 X lo-” M). IV. AcPyAD (6 X 10m5M). Curve B. Equilibrium value. X,, 341 rnp. Sample beam: I. LDH (3 x 10m6~), AcPyAD (6 X lo-&~), pyruvate (10e3M). II. Buffer. Reference beam: III. LDH (3 X 10m6M). IV. AcPyAD (6 X 10e5 M), pyruvate (1Om3M).
LACTATE
DEHYDROGENASE-COENZYME
COMPLEXES
571
NADH. The appropriate control experiments are reported in each case. I. BINARY AND ABORTIVE TERNARY COMPLEXES INVOLVING AcPyAD In Fig. 1 spectral curves for LDH, AcPyAD and pyruvate vs. buffer in the region 300-500 rnp are shown for reference. In each case the absorbance above 300 rnr is relatively low. Concentrations and exact conditions are described in the legend of this and succeeding figures. The difference spectra in Fig. 2 are those which result when a mole ratio of l/20 for LDH/AcPyAD is established. Other mole ratios were investigated (from l/10 to l/160). The absorbance of the binary LDH . AcPyAD complexes increased with decreasing mole ratios at constant LDH concentration. A decision to use a l/20 mole ratio for this series was made. The observed absorbance peak for the binary complex (X,, = 355 rnp) shows an extinction which is about l/8 of that which would be expected (and was found; see Section 3) if AcPyAD is replaced by AcPyADH at an equal concentration. The x,, of 355 rnp is clearly different from that for either free (363 mp) or enzyme-bound AcPyADH (361 rnp) . When pyruvate was added to a concentration of lo-” M, the resulting difference spectrum was that of the abortive ternary complex (mole ratio: LDH/AcPyAD/pyruvate, l/20/333) with a X,,, of 341 mM. When the pyruvate concentration was varied, the extinction was found to increase with increasing pyruvate concentration up to 6 X lo-” M. Measurements were not made at higher pyruvate concentrations. When it was noted that the 341 m/l absorbance increased slowly with time, a study was made of the sequence of events. Upon the addition of pyruvate at zero time, the 355 rnw absorbance fell rapidly during the first few seconds. This was followed by a slow increase of the 341 rnw absorbance to an equilibrium value during some 50 minutes, as shown in Fig. 3. The observed nonlinear rate has been confirmed in five separate experiments.
FIG. 3. Time dependence of spectral changes associated with abortive ternary complex formation. A&U “,,, vs. Time (minutes). Continued observation of system of Curve B, Fig. 2.
It was of immediate interest to note whether the observed spectral changes which seemed so remarkably slow were reversible on dialysis against AcPyADbuffer. Upon such dialysis at 0” for 20 hours against three changes of 80 volumes each of dialyzing medium, the original binary complex reestablished itself spectrally. When the LDH was assayed under comparative conditions, the enzyme was found to be fully active, just as if it had not experienced the rigors of abortive ternary complex formation and prolonged dialysis. It should be remembered that kinetic measurements of LDH activity with AcPyAD as coenzyme at pH 8.6 are subject to the limitation of lactate (substrate) inhibition, a result which is not observed when NAD is coenzyme (2). In further studies of the slow spectral changes just reported, it was found that there was no effect of continuous illumination during the process and that there were no spectral changes induced by pyruvate alone or by interaction of pyruvate and AcPyAD prior to ternary complex formation. Further, it was found that pyruvate could be added before AcPyAD with no different effect. The LDH was demonstrated to be completely stable for the periods of time involved. In order to test the specificity requirement for pyruvate of spectral changes accompanying abortive ternary complexation, an experiment was carried out in which cr-ketobutyrate was substituted for
VESTLING
AND
apparent Michaelis constant for cY-ketobutyrate as compared to pyruvate (2). Dialysis vs. AcPyAD-buffer led to the original spectrum of the binary complex, and the enzyme activity was fully recovered.
5
:
3&,
0 II 3oc I
KONSCH
350 400 WIVELENGTH
450 (mG1
FIG. 4. Binary and ternary systems involving NAD. Difference spectra, double tandem system, pH 7.8, 2P. Curve A. h, 335 mp. Sample beam: I. LDH (1.2 X 10e5 M), NAD (4.8 X lo-’ M). II. Buffer. Reference beam: III. LDH (1.2 X 10e5ru). IV. NAD (4.8 X lo-’ M). Curve B. Equilibrium value. A, 323 mp. Sample beam: I. LDH (1.2 X 10s5 M), NAD (4.8 X lo-’ M), pyruvate (10m3 M). II. Buffer. Reference beam: III. LDH (1.2 X 10m5 M). IV. NAD (4.8 X 10m4 M), pyruvate (10m3 M).
II. BINARY AND ABORTIVE TERNARY COMPLEXES INVOLVING NAD The binary complex, LDH-NAD (mole ratio, l/40) exhibits a very low absorbance peak with a x,, of 335 rnp, as shown in Fig. 4. In this experiment four times the LDH concentration and eight times the NAD concentration were used as compared to the studies with AcPyAD (Fig. 2). When pyruvate was added to a final concentration of 10e3 M, the spectrum of the abortive ternary complex resulted as shown in Fig. 4. In this case a time dependence of the development of the 323 rnp peak was again observed. Upon addition of pyruvate, the 335 rnk absorbance fell for a few seconds, and the 323 rng absorbance then rose slowly during some four hours to an equilibrium value @,,,= of 323 mp). In three separate experiments very similar rates of spectral change were observed (Fig. 5). There was no effect of continued or interrupted illumination. However, the spectral changes were not reversible on prolonged dialysis (146 hours) vs. NAD-buffer. The original
I 0
I TIM
2 (Hours)
3
4
5
5. Time dependence of spectral changes associated with abortive ternary complex formation. AG2am# vs. Time (hours). Continued observation of Curve B, Fig. 4. FIG.
pyruvate, all other conditions being those described in Fig. 2. An absorbance peak with a X,, of 346 rnr developed during about 45 minutes, and the peak height was about 517 of that for the ternary pyruvate . abortive complex involving This result is consistent with the slower maximum catalytic rate and the higher
% 5 L” O-092. : *
-0.04300
350
400 WAVELENQTH
450
500
(mu)
Frc. 6. Binary system involving AcPyADH. Difference spectrum, double tandem system, pH 7.8, 25”. Sample beam: I. LDH (3 X 10m6M), AcF’yADH (6 X 10d5~). II. Buffer. Reference beam: III. LDH (3 X 1O-6 M). IV. AcPyADH (6 X 1O-5 M).
LACTATE
DEHYDROGENASE-COENZYME
spectrum of the binary complex was not recovered, but the enzyme was fully active. Control experiments showed no interaction between NAD and pyruvate, and the LDH was fully stable both in the presence and absence of NAD during the 146-hour period. Thus, the effects observed with AcPyAD and NAD were somewhat parallel, the principal differences being the greater absorbance of the complexes involving the coenzyme analog and the failure to reestablish the original binary complex spectrum upon prolonged dialysis of LDH . NAD . pyruvate to remove pyruvate.
COMPLEXES
573
then subtracts the curves. The curve for free AcPyADH shows a X,, at 363 rnp, while that for LDH-bound AcPyADH shows a X,, at 361 rnp. When D, L-lactate was added to the binary complex to a final concentration of 0.05 M, no spectral change consistent with the formation of an abortive ternary complex was noted.
IV. BINARY COMPLEXES INVOLVING NADH When the binary complex LDH . NADH was studied at an LDH concentration twice and a NADH concentration four times those used in the experiments III. BINARY AND TERNARY COMPLEXES with AcPyADH, the curve shown in Fig. INVOLVING AcPyADH 7 was obtained. A slow spectral change during 22 hours Partly for the sake of comparison, a series of studies involving reduced co- accompanied the formation of the binary enzyme or reduced coenzyme analog was complex. The depth of the trough inundertaken. In Fig. 6, a difference spec- creased from an absorbance of 0.02 to 0.1, trum is shown to illustrate the effect of and the position of the Xminshifted from binding of AcPyADH to LDH at a mole 355 to 340 rnp. This result was unexpected ratio of 1 LDH to 20 AcPyADH. The peak and requires some explanation. It was at 333 rnp and the trough at 387 rnp likely that one was observing an endemonstrate the small differences which hanced instability of NADH when a are less obvious but clearly discernible fraction of it was bound to LDH. When when one traces the spectra directly and lactate was added, as in the experiment of Fig. 6, no spectral shift was observed. DISCUSSION
-0.10
t ,y, ’ ’ ’ 350 300
I I 400
WAVELENGTH
, 450
, I. 500
tmG)
FIG. 7. Binary system involving NADH. Difference spectra, double tandem system, pH 7.8, 25O. Time dependence of interaction and shift of h,,. . Sample beam: I. LDH (6 X 1O-6~), NADH (2.4 X IO-” M). II. Buffer. Reference beam: III: LDH (6 X lo-” M). IV. NADH (2.4 X lo-’ M). Times after mixing: Curve A, 5 min, h,,, 355 mp; Curve B, 1 hour, A,,,,. 348 rnw; Curve C, 6 hours, A,,, 342 mp; Curve D, 22 hours, x,~. 341 mp.
It is useful to have a structural model which will serve as a basis for an interpretation of the results which have been presented. Such a model must be consistent not only with spectral observations but also with fluorescence and kinetic data. One of the authors of this paper proposed such a model earlier (27); it is reproduced here in slightly modified form in Fig. 8. Kinetic data from several laboratories are consistent with a compulsory binding sequence mechanism for pyridine nucleotide-linked dehydrogenases (11, 13, 16, 20, 23, 24, 29, 30). In such a mechanism the first step is the binding of coenzyme, a process which is now seen to involve marked spectral changes in the case of the oxidized coenzyme and which presumably also involves conformational
574
VESTLING
AND KijNSCH
FIG. 8. Hypothetical Structural Model, LDH Reaction Site.
adjustments within the enzyme of such a nature as to facilitate the formation of a reactive ternary complex when the substrate is added. The act of binding the oxidized coenzyme or analog leads to the spectral changes shown in this report and is postulated to involve partial hybridization of the substituted pyridine ring so that the catalytic act is one of facilitating hydride ion shift to and from the partial carbonium ion at position 4, as depicted in Fig. 8. The observed spectral development when NAD or AcPyAD is bound to LDH is consistent with the existence of some “quinoid structure” in the pyridine ring. That this behavior cannot be explained by assuming that a fraction of the bound oxidized coenzyme is reduced in the binding process seems clear from a study of the spectral behavior of the bound reduced coenzymes. When the spectral changes accompanying the binding of oxidized coenzymes were first observed, the natural question was to ask whether small amounts of trapped lactate or other substrate in the enzyme were reacting to produce some reduced coenzyme. Ln the experiments reported here the LDH was purified to maximum specific activity, and the possibility of lactate contamination seems remote. Further-in experiments involving binary complexes of LDH . reduced coenzyme-the addition of pyruvate led promptly to the disappearance of the bound reduced coenzyme spectrum and the appearance of the abortive ternary complex spectrum. In Fig. 8 the suggestion is made that a neighboring SH group may serve to stabilize the LDH-oxidized coenzyme
complex. This is a speculation only, although evidence on this point is being sought. The most interesting and unexpected result reported in this paper is that of the very slow spectral change which accompanies the formation of abortive ternary complexes. One should remember that pyruvate is a potent inhibitor of the LDH-catalyzed system from both sides of the reaction. Pyruvate inhibition from the pyruvate side is markedly pH-dependent (4, 13). Thus pyruvate will apparently compete with lactate in the formation of ternary complexes, but it appears that pyruvate must also be considered to bind elsewhere to the enzyme-oxidized coenzyme complex in an allosteric fashion. It is the conformational adjustments accompanying this pyruvate binding which are exceedingly slow at 25” and of such a nature as to influence the electronic environment of the pyridine chromophore of the bound oxidized coenzyme. The fact that the spectral changes are completely reversible when AcPyAD is involved, and not reversible when NAD is involved indicates an important difference in the interactions at the active site. In both cases enzyme activity was fully recovered following prolonged dialysis against oxidized coenzyme-buffer to remove pyruvate. The recent report by Gutfreund et al. (12) provides substantiating evidence with respect to the abortive ternary complex, pig heart LDH . NAD . pyruvate, formed at pH 6. Considerably higher concentrations of pyruvate (0.03 M) were used by these investigators, and very much shorter periods of time were required for the development of the 325 rnw absorbance peak. Gutfreund et al. noted that increasing the pyruvate concentration in their systems at constant NAD concentration did not result in a saturating rate of development of 325 ml absorbance. These authors also studied fluorescence associated with abortive ternary complex formation and found these changes to be reversible on dilution
LACTATE
DEHYDROGENASE-COENZYME
of LDH . NAD . pyruvate with NAD . buffer. They suggested that the pyruvatebinding site for enzymatic reduction of pyruvate is not involved in formation of the abortive ternary complex on the basis of their demonstration that oxamate, a competitive inhibitor for pyruvate reduction, did not influence the rate of formation of the ternary complex. The results of the investigations described in the present paper also suggest that pyruvate is acting in an allosteric manner. REFERENCES
14. 15.
16. 17.
18.
1. ANDERSON, S. R., unpublished results. 2. ANDERSON, S. R., FLORINI, J. R., AND VESTLING, C. S., J. Biol. Chem. 239, 2991(1964). 3. BAKER, R. H., Biochemistry 1, 41 (1962). 4. BAPTIST, J. N., PH.D. Thesis, University of Illinois (1957). 5. BEILSTEIN HANDBUCH, Second Supplement, Vol. 1, p. 523. 6. CHANCE, B., AND NEILANDS, J. B., J. Biol. Chem. 199, 383 (1952). 7. C~orn, M. M., AND KAPLAN, N. O., in “Methods in Enzymology” S. P. Colowick, and N. 0. Kaplan, eds., Vol. 3, 891. Academic Press, New York, (1957). 8. DAVISSON, E. O., GIBSON, D. M., RAY, B. R., AND VESTLING, C. S., J. Phys. Chem. 57, 609 (1953). 9. FISHER, H.
13.
19. 20. 21. 22. 23. 24. 25. 26. 27.
F.,
AND CROSS, D.
153, 414 (1966). 10. FROMM, H. J., Biochim.
Biophys.
G.,
Science
Acta 52, 199
28.
(1961). 11. FROMM, H. J., AND NELSON, D. R., J. Biol.
29.
Chem. 237, 215 (1962). 12. GUTFREUND, H., CANTWELL, R.,
30. MCMLJRRAY,
COMPLEXES
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C. H. CRIDDLE, R. S., AND HATHAWAY, G., Biochem. J., 106,683 (1968). HAKALA, M. T., GLAID, A. J., AND SCHWERT, G. W., J. Biol. Chem. 221, 191 (1956). HSIEH, W. T., AND VESTLING, C. S., Biochem. Prepamtions 11, 69 (1966). KAPLAN, N. O., CIOTTI, M. M., AND STOLZENBACH, F. E., Arch. Biochem. Biophys. 69, 441(1957). NYGAARD, A. P., AND THEORELL, H., Acta Chem. Scand. 9, 1300 (1955). OSER, B. L., Editor, Hawk’s Physiological Chemistry McGraw-Hill, New York, 14th Edition, p. 1243 (1965). PALIT, S. R., Ind. Eng. Chem., Anal. Ed. 18, 246 (1946). PFLEIDERER, G., AND HOHNHOLZ, E., Biochem. Z. 331, 245 (1959). RAVAL, D. N., AND WOLFE, R. G., Biochemistry 1, 1112 (1962). STEGINK, L. D., AND VESTLING, C. S., J. Biol. Chem. 241,4923 (1966). THEORELL, H., AND BONNICHSEN, R., Acta Chem. &and. 5, 1105 (1951). THEORELL, H., NYGAARD, A. P., AND BONNICHSEN, R., Acta Chem. &and. 9, 1148 (1955). THEORELL, H., AND WINER, A. D., Arch. Biothem. Biophys. 83,291(1959). THEOFELL, H., AND YONETANI, T., Biochem. z. 338, 537 (1963). THEORELL, H., AND YONETANI, T., Arch. Biothem. Biophys. 106, 252 (1964). VESTLING, C. S. in D. Glick, Editor, “Methods of Biochemical Analysis,” Interscience Publishers, Inc., New York, Vol. X, p. 137 (1962). WINER, A. D., Acta Chem. Scared. 17, Suppl. 1, 203( 1963). WRAT~EN, C. C., AND CLELAND, W. W., Biochemistry 2, 935 (1963). ZEWE, V., AND FROMM. H. J., J. Biol. Chem. 237, 1666 (1962).
AND BIOPHYSICS
127, 566-575 (196%
Spectrophotometric
Analysis
Dehydrogenase-Coenzyme Analog
of Biochemistry,
and Coenzyme
Complexes
CARL S. VESTLING Department
of Rat Liver Lactate
AND
University
1
ULRICH of Iowa,
Iowa
KUNSCH City, Iowa 52240
Received March 22, 1968; accepted May 11, 1968 A study of difference spectra in the range, 300-500 rnr is reported involving highly purified rat liver lactate dehydrogenase (LDH) and oxidized or reduced coenzyme or coenzyme analog (nicotinamide-adenine dinucleotide (NAD) or acetylpyridine-adenine dinucleotide ( AcPy AD). The formation of the binary complexes, LDH . NAD or LDH AcPyAD is associated with the development of spectral bands with A,,, at 335 rnr and 355 rnp, respectively. The observed absorbance is proportional to coenzyme concentration at fixed LDH concentration. The spectral behavior of the LDH .oxidized coenzyme binary complexes is clearly distinguishable from that of the binary complexes involving NADH or AcPyADH. A conclusion is that the binding of oxidized coenzymes to LDH results in a significant change in the chromophoric behavior of a hybridized form of the substituted pyridine ring. The formation of abortive ternary complexes involving LDH . oxidized coenzyme pyruvate or LDH oxidized coenzyme . a-ketobutyrate was accompanied by slow progressive spectral changes which reached equilibrium values, dependent on the concentration of all three reactants. For LDH NAD pyruvate A- was 323 mr; for LDH AcPyAD pyruvate A, was 341 mp. The spectral changes were fully reversible upon dialysis vs. AcPyAD-buffer in the one case but not upon dialysis vs. NAD-buffer in the other. Complete recovery of enzyme activity was noted in each case after dialysis to remove pyruvate or ol-ketobutyrate. The spectral changes are considered to be those associated with the allcsteric binding of pyruvate or a-ketobutyrate to binary complexes of LDH . oxidized coenzyme with re sulting conformational changes in LDH which affect the active sites. No abortive ternary complex involving LDH-reduced coenzyme (or reduced coenzyme analog) -lactate could be detected by spectral means.
Spectral studies from various laboratories have produced reports of enzyme-reduced coenzyme (or reduced coenzyme analog) complexes (6, 9, 15, 19, 22, 25, 26). These studies involved the measurement of difference spectra in the case of several dehydrogenases and indicated relatively small spectral shifts when free reduced coenzyme was compared to bound coenzyme (spectral reduced
range: 300400 my). The interpretation of such experiments has generally been that the act of binding of reduced coenzyme exerted a relatively minor influence on the chromophoric behavior of the dihydropyridine residue of the molecule. On the other hand there has appeared only one preliminary report, which the present authors have noted, of spectral studies of the effect of binding of oxidized coenzyme (or analog) to a dehydrogenase (28). In this case the author described a very small spectral change when bovine
’ This work was supported by Grant CA 07617 from the National Cancer Institute, National Institutes of Health. 568
LACTATE
DEHYDROGENASE-COENZYME
heart lactate dehydrogenase (LDH)’ was combined with NAD, but a very marked change when the enzyme was combined with the acetyl pyridine analog of NAD (AcPyAD). Winer (28) reported a X,,, for the binary complex, LDH . AcPyAD, of 355 rnp at pH 8.15. The experiments which are described by the present authors serve to confirm and extend Winer’s observations and lend themselves to the interpretation that the act of binding of AcPyAD or NAD to LDH is one in which there is a decided influence of the enzyme on the chromophoric system of the oxidized coenzyme or analog (which in the free state show very little absorbance in the spectral region, 300400 mp). Fromm and co-workers (10, 11, 19) were the first to call attention to “abortive” ternary complexes formed from enzyme, coenzyme and “wrong substrate” (ex., LDH . NAD . pyruvate). Winer (28) reported a similar result and indicated that the X, for the abortive ternary complex formed from bovine heart LDH, NAD, and pyruvate was 325 rnp under the same conditions mentioned above. Recently Gutfreund et al. (12) reported the formation of pig heart LDH . NAD + pyruvate complexes in a study of the inhibition of LDH by pyruvate. They measured increases in absorbance at 325 rnr and correlated spectral changes with fluorescence quenching and inhibition. The observations of Gutfreund et al. lend support to the results by the present authors. Anderson (1) performed some preliminary experiments involving rat liver LDH, NAD or AcPyAD and pyruvate and laid the foundation for the present study. Anderson, Florini and Vestling (2) interpreted their kinetic measurements on the basis of abortive ternary complexation of LDH . NAD . pyruvate but could demonstrate no such effect ‘Abbreviations: LDH, Rat liver lactate dehydrogenase; NAD, NADH, Nicotinamide adenine dioxidized nucleotide, and reduced; AcPyAD, AcPyADH, Acetylpyridine adenine dinucleotide, oxidized and reduced. A, Absorbance.
569
COMPLEXES
with the components: LDH . NADH . lactate. In the present studies a series of spectral determinations is described and a plausible interpretation proposed. In addition, an unusual time dependence has been found for spectral changes accompanying the formation of abortive ternary complexes. These spectral changes are fully reversible in the case of AcPyAD, but not in the case of NAD. No loss of enzyme activity is encountered after dialysis to remove the “wrong substrate” in either case. A time dependence has also been noted for spectral changes accompanying binary complex formation when NADH but not AcPyADH is involved. EXPERIMENTAL
PROCEDURES
RAT LIVER LACTATE DEHYDROGENASE Rat liver LDH was isolated, purified and assayed according to Hsieh and Vestling (14). The enzyme used in these experiments had a specific activity of 100 f 4 I.U./mg of protein, which value corresponds to that for maximally pure enzyme. This LDH is a tetramer of the M, type (21) with a molecular weight of 126,000 (8). Before each experiment 1.6 ml of LDH solution was dialyzed at 0’ under N2 against 120 ml of a 0.02 M potassium phosphate buffer, pH 7.8, which was 0.02 M with respect to ammonium sulfate and 10.” M with respect to 2-mercaptoethanol. Five changes of buffer were made during a 4-hour period. This buffer was used for all spectrophotometric determinations. At the end of the dialysis aliquots were taken out and assayed. The protein concentration was calculated from the absorbance at 280 rnr = 12.6) and from the measurement of en(E:%, zymatic activity [l mg of pure LDH is equivalent to 101.5 International Units (rmoles NADH ml-’ mini’].
COENZYMES The following materials were analyzed according to Ciotti and Kaplan (7). 1. NAD: /%NAD. 3.5 HzO, 987, pure, Sigma Chemical Company, found to be 97-98:;’ pure. 2. AcPyAD: AcPyAD ‘2 HZO, 91”; pure, P.L. Biochemicals, Inc., found to be 90% pure. 3. NADH: fl-Na?NADH .3 H20, 98% pure, Sigma Chemical Company, found to be 91% pure.
570
VESTLING
-----Sam Bea --uI II
AND KijNSCH
4. AcPyADH: BaAcPyAD .4 Hz0 prepared in this laboratory by reduction of 91% pure AcPyAD . 2 Hz0 (P.L. Biochemicals, Inc.) with yeast ADH, and found to be 83% pure. 5. NasAcPyADH. 2Hz0, 93% pure (P.L. Biochemicals, Inc.), was found to be 86% pure.
II
SUBSTRATES 1. Lactate: Lithium D, L-lactate, anhydr., was prepared in this laboratory from 85% pure D, Llactic acid (17) and found to be 97% pure in a potentiometric titration in an organic solvent mixture (18). 2. Pyruvate: Na-pyruvate, 99% pure, Sigma Chemical Company.
--
Reference
Beam
1
SCHEME
OTHER COMPOUNDS P-Mercaptoethanol (Eastman) was redistilled in uacuo (bp 57-58”, 14 mm). The resulting vg was 1.5011 as compared to the value of 1.4996 (5). It was stored under nitrogen in l-ml ampoules. Dialysis tubing: Visking, lo-mm diameter, flat. Sections were placed in boiling distilled water during two 30-minute periods, to remove ultraviolet absorbing material. All other materials used were of analytical reagent grade.
WAVELENGTH
(mu)
FIG. 1. Difference spectra. Curve A, LDH (3 x 10m6M) vs. buffer. Curve B, AcPyAD (6 X 1O-5 M) vs. buffer. Curve C. sodium pyruvate (10-j M) vs. buffer.
SPECTROPHOTOMETRIC PROCEDURE A double tandem system of matched cuvettes was used in a Cary Model 15 recording spectrophotometer (cuvette volume, 1.5 ml; light path, 1.0 cm). The cuvette arrangement is sketched in Scheme 1. The cuvette numbers will appear in the legends of the appropriate figures. The cuvette positioning and alignment were carefully checked. All spectral measurements were made at 25 f 1’. Constant temperature was maintained by circulation of water through the jacketed cuvette compartment of the spectrophotometer. The cuvettes used were closed with discs cut from gum rubber “serum caps” attached with gum rubber sleeves. Measured volumes of appropriate solutions were added through the caps from calibrated syringes. The final volume in each cuvette was 1.0 ml for all binary complex systems. Additions of 10 ~1 were made to form the ternary complexes; the resulting volume changes . were within l?; and were neglected. RESULTS
The data to be presented are grouped in four sections. Complexation involving AcPyAD precedes that of NAD, and that involving AcPyADH precedes that of
A
X,OX.
WAVELENGTH
33Bmu
(mu)
FIG. 2. Binary and ternary systems involving AcPyAD. Difference spectra, double tandem system, pH 7.8, 25”. Curve A. A,, 355 mp. Sample beam: I. LDH(3 X 10-"M), ACP~AD(~ X lo-"M).II. Buffer. Reference beam: III. LDH (3 X lo-” M). IV. AcPyAD (6 X 10m5M). Curve B. Equilibrium value. X,, 341 rnp. Sample beam: I. LDH (3 x 10m6~), AcPyAD (6 X lo-&~), pyruvate (10e3M). II. Buffer. Reference beam: III. LDH (3 X 10m6M). IV. AcPyAD (6 X 10e5 M), pyruvate (1Om3M).
LACTATE
DEHYDROGENASE-COENZYME
COMPLEXES
571
NADH. The appropriate control experiments are reported in each case. I. BINARY AND ABORTIVE TERNARY COMPLEXES INVOLVING AcPyAD In Fig. 1 spectral curves for LDH, AcPyAD and pyruvate vs. buffer in the region 300-500 rnp are shown for reference. In each case the absorbance above 300 rnr is relatively low. Concentrations and exact conditions are described in the legend of this and succeeding figures. The difference spectra in Fig. 2 are those which result when a mole ratio of l/20 for LDH/AcPyAD is established. Other mole ratios were investigated (from l/10 to l/160). The absorbance of the binary LDH . AcPyAD complexes increased with decreasing mole ratios at constant LDH concentration. A decision to use a l/20 mole ratio for this series was made. The observed absorbance peak for the binary complex (X,, = 355 rnp) shows an extinction which is about l/8 of that which would be expected (and was found; see Section 3) if AcPyAD is replaced by AcPyADH at an equal concentration. The x,, of 355 rnp is clearly different from that for either free (363 mp) or enzyme-bound AcPyADH (361 rnp) . When pyruvate was added to a concentration of lo-” M, the resulting difference spectrum was that of the abortive ternary complex (mole ratio: LDH/AcPyAD/pyruvate, l/20/333) with a X,,, of 341 mM. When the pyruvate concentration was varied, the extinction was found to increase with increasing pyruvate concentration up to 6 X lo-” M. Measurements were not made at higher pyruvate concentrations. When it was noted that the 341 m/l absorbance increased slowly with time, a study was made of the sequence of events. Upon the addition of pyruvate at zero time, the 355 rnw absorbance fell rapidly during the first few seconds. This was followed by a slow increase of the 341 rnw absorbance to an equilibrium value during some 50 minutes, as shown in Fig. 3. The observed nonlinear rate has been confirmed in five separate experiments.
FIG. 3. Time dependence of spectral changes associated with abortive ternary complex formation. A&U “,,, vs. Time (minutes). Continued observation of system of Curve B, Fig. 2.
It was of immediate interest to note whether the observed spectral changes which seemed so remarkably slow were reversible on dialysis against AcPyADbuffer. Upon such dialysis at 0” for 20 hours against three changes of 80 volumes each of dialyzing medium, the original binary complex reestablished itself spectrally. When the LDH was assayed under comparative conditions, the enzyme was found to be fully active, just as if it had not experienced the rigors of abortive ternary complex formation and prolonged dialysis. It should be remembered that kinetic measurements of LDH activity with AcPyAD as coenzyme at pH 8.6 are subject to the limitation of lactate (substrate) inhibition, a result which is not observed when NAD is coenzyme (2). In further studies of the slow spectral changes just reported, it was found that there was no effect of continuous illumination during the process and that there were no spectral changes induced by pyruvate alone or by interaction of pyruvate and AcPyAD prior to ternary complex formation. Further, it was found that pyruvate could be added before AcPyAD with no different effect. The LDH was demonstrated to be completely stable for the periods of time involved. In order to test the specificity requirement for pyruvate of spectral changes accompanying abortive ternary complexation, an experiment was carried out in which cr-ketobutyrate was substituted for
VESTLING
AND
apparent Michaelis constant for cY-ketobutyrate as compared to pyruvate (2). Dialysis vs. AcPyAD-buffer led to the original spectrum of the binary complex, and the enzyme activity was fully recovered.
5
:
3&,
0 II 3oc I
KONSCH
350 400 WIVELENGTH
450 (mG1
FIG. 4. Binary and ternary systems involving NAD. Difference spectra, double tandem system, pH 7.8, 2P. Curve A. h, 335 mp. Sample beam: I. LDH (1.2 X 10e5 M), NAD (4.8 X lo-’ M). II. Buffer. Reference beam: III. LDH (1.2 X 10e5ru). IV. NAD (4.8 X lo-’ M). Curve B. Equilibrium value. A, 323 mp. Sample beam: I. LDH (1.2 X 10s5 M), NAD (4.8 X lo-’ M), pyruvate (10m3 M). II. Buffer. Reference beam: III. LDH (1.2 X 10m5 M). IV. NAD (4.8 X 10m4 M), pyruvate (10m3 M).
II. BINARY AND ABORTIVE TERNARY COMPLEXES INVOLVING NAD The binary complex, LDH-NAD (mole ratio, l/40) exhibits a very low absorbance peak with a x,, of 335 rnp, as shown in Fig. 4. In this experiment four times the LDH concentration and eight times the NAD concentration were used as compared to the studies with AcPyAD (Fig. 2). When pyruvate was added to a final concentration of 10e3 M, the spectrum of the abortive ternary complex resulted as shown in Fig. 4. In this case a time dependence of the development of the 323 rnp peak was again observed. Upon addition of pyruvate, the 335 rnk absorbance fell for a few seconds, and the 323 rng absorbance then rose slowly during some four hours to an equilibrium value @,,,= of 323 mp). In three separate experiments very similar rates of spectral change were observed (Fig. 5). There was no effect of continued or interrupted illumination. However, the spectral changes were not reversible on prolonged dialysis (146 hours) vs. NAD-buffer. The original
I 0
I TIM
2 (Hours)
3
4
5
5. Time dependence of spectral changes associated with abortive ternary complex formation. AG2am# vs. Time (hours). Continued observation of Curve B, Fig. 4. FIG.
pyruvate, all other conditions being those described in Fig. 2. An absorbance peak with a X,, of 346 rnr developed during about 45 minutes, and the peak height was about 517 of that for the ternary pyruvate . abortive complex involving This result is consistent with the slower maximum catalytic rate and the higher
% 5 L” O-092. : *
-0.04300
350
400 WAVELENQTH
450
500
(mu)
Frc. 6. Binary system involving AcPyADH. Difference spectrum, double tandem system, pH 7.8, 25”. Sample beam: I. LDH (3 X 10m6M), AcF’yADH (6 X 10d5~). II. Buffer. Reference beam: III. LDH (3 X 1O-6 M). IV. AcPyADH (6 X 1O-5 M).
LACTATE
DEHYDROGENASE-COENZYME
spectrum of the binary complex was not recovered, but the enzyme was fully active. Control experiments showed no interaction between NAD and pyruvate, and the LDH was fully stable both in the presence and absence of NAD during the 146-hour period. Thus, the effects observed with AcPyAD and NAD were somewhat parallel, the principal differences being the greater absorbance of the complexes involving the coenzyme analog and the failure to reestablish the original binary complex spectrum upon prolonged dialysis of LDH . NAD . pyruvate to remove pyruvate.
COMPLEXES
573
then subtracts the curves. The curve for free AcPyADH shows a X,, at 363 rnp, while that for LDH-bound AcPyADH shows a X,, at 361 rnp. When D, L-lactate was added to the binary complex to a final concentration of 0.05 M, no spectral change consistent with the formation of an abortive ternary complex was noted.
IV. BINARY COMPLEXES INVOLVING NADH When the binary complex LDH . NADH was studied at an LDH concentration twice and a NADH concentration four times those used in the experiments III. BINARY AND TERNARY COMPLEXES with AcPyADH, the curve shown in Fig. INVOLVING AcPyADH 7 was obtained. A slow spectral change during 22 hours Partly for the sake of comparison, a series of studies involving reduced co- accompanied the formation of the binary enzyme or reduced coenzyme analog was complex. The depth of the trough inundertaken. In Fig. 6, a difference spec- creased from an absorbance of 0.02 to 0.1, trum is shown to illustrate the effect of and the position of the Xminshifted from binding of AcPyADH to LDH at a mole 355 to 340 rnp. This result was unexpected ratio of 1 LDH to 20 AcPyADH. The peak and requires some explanation. It was at 333 rnp and the trough at 387 rnp likely that one was observing an endemonstrate the small differences which hanced instability of NADH when a are less obvious but clearly discernible fraction of it was bound to LDH. When when one traces the spectra directly and lactate was added, as in the experiment of Fig. 6, no spectral shift was observed. DISCUSSION
-0.10
t ,y, ’ ’ ’ 350 300
I I 400
WAVELENGTH
, 450
, I. 500
tmG)
FIG. 7. Binary system involving NADH. Difference spectra, double tandem system, pH 7.8, 25O. Time dependence of interaction and shift of h,,. . Sample beam: I. LDH (6 X 1O-6~), NADH (2.4 X IO-” M). II. Buffer. Reference beam: III: LDH (6 X lo-” M). IV. NADH (2.4 X lo-’ M). Times after mixing: Curve A, 5 min, h,,, 355 mp; Curve B, 1 hour, A,,,,. 348 rnw; Curve C, 6 hours, A,,, 342 mp; Curve D, 22 hours, x,~. 341 mp.
It is useful to have a structural model which will serve as a basis for an interpretation of the results which have been presented. Such a model must be consistent not only with spectral observations but also with fluorescence and kinetic data. One of the authors of this paper proposed such a model earlier (27); it is reproduced here in slightly modified form in Fig. 8. Kinetic data from several laboratories are consistent with a compulsory binding sequence mechanism for pyridine nucleotide-linked dehydrogenases (11, 13, 16, 20, 23, 24, 29, 30). In such a mechanism the first step is the binding of coenzyme, a process which is now seen to involve marked spectral changes in the case of the oxidized coenzyme and which presumably also involves conformational
574
VESTLING
AND KijNSCH
FIG. 8. Hypothetical Structural Model, LDH Reaction Site.
adjustments within the enzyme of such a nature as to facilitate the formation of a reactive ternary complex when the substrate is added. The act of binding the oxidized coenzyme or analog leads to the spectral changes shown in this report and is postulated to involve partial hybridization of the substituted pyridine ring so that the catalytic act is one of facilitating hydride ion shift to and from the partial carbonium ion at position 4, as depicted in Fig. 8. The observed spectral development when NAD or AcPyAD is bound to LDH is consistent with the existence of some “quinoid structure” in the pyridine ring. That this behavior cannot be explained by assuming that a fraction of the bound oxidized coenzyme is reduced in the binding process seems clear from a study of the spectral behavior of the bound reduced coenzymes. When the spectral changes accompanying the binding of oxidized coenzymes were first observed, the natural question was to ask whether small amounts of trapped lactate or other substrate in the enzyme were reacting to produce some reduced coenzyme. Ln the experiments reported here the LDH was purified to maximum specific activity, and the possibility of lactate contamination seems remote. Further-in experiments involving binary complexes of LDH . reduced coenzyme-the addition of pyruvate led promptly to the disappearance of the bound reduced coenzyme spectrum and the appearance of the abortive ternary complex spectrum. In Fig. 8 the suggestion is made that a neighboring SH group may serve to stabilize the LDH-oxidized coenzyme
complex. This is a speculation only, although evidence on this point is being sought. The most interesting and unexpected result reported in this paper is that of the very slow spectral change which accompanies the formation of abortive ternary complexes. One should remember that pyruvate is a potent inhibitor of the LDH-catalyzed system from both sides of the reaction. Pyruvate inhibition from the pyruvate side is markedly pH-dependent (4, 13). Thus pyruvate will apparently compete with lactate in the formation of ternary complexes, but it appears that pyruvate must also be considered to bind elsewhere to the enzyme-oxidized coenzyme complex in an allosteric fashion. It is the conformational adjustments accompanying this pyruvate binding which are exceedingly slow at 25” and of such a nature as to influence the electronic environment of the pyridine chromophore of the bound oxidized coenzyme. The fact that the spectral changes are completely reversible when AcPyAD is involved, and not reversible when NAD is involved indicates an important difference in the interactions at the active site. In both cases enzyme activity was fully recovered following prolonged dialysis against oxidized coenzyme-buffer to remove pyruvate. The recent report by Gutfreund et al. (12) provides substantiating evidence with respect to the abortive ternary complex, pig heart LDH . NAD . pyruvate, formed at pH 6. Considerably higher concentrations of pyruvate (0.03 M) were used by these investigators, and very much shorter periods of time were required for the development of the 325 rnw absorbance peak. Gutfreund et al. noted that increasing the pyruvate concentration in their systems at constant NAD concentration did not result in a saturating rate of development of 325 ml absorbance. These authors also studied fluorescence associated with abortive ternary complex formation and found these changes to be reversible on dilution
LACTATE
DEHYDROGENASE-COENZYME
of LDH . NAD . pyruvate with NAD . buffer. They suggested that the pyruvatebinding site for enzymatic reduction of pyruvate is not involved in formation of the abortive ternary complex on the basis of their demonstration that oxamate, a competitive inhibitor for pyruvate reduction, did not influence the rate of formation of the ternary complex. The results of the investigations described in the present paper also suggest that pyruvate is acting in an allosteric manner. REFERENCES
14. 15.
16. 17.
18.
1. ANDERSON, S. R., unpublished results. 2. ANDERSON, S. R., FLORINI, J. R., AND VESTLING, C. S., J. Biol. Chem. 239, 2991(1964). 3. BAKER, R. H., Biochemistry 1, 41 (1962). 4. BAPTIST, J. N., PH.D. Thesis, University of Illinois (1957). 5. BEILSTEIN HANDBUCH, Second Supplement, Vol. 1, p. 523. 6. CHANCE, B., AND NEILANDS, J. B., J. Biol. Chem. 199, 383 (1952). 7. C~orn, M. M., AND KAPLAN, N. O., in “Methods in Enzymology” S. P. Colowick, and N. 0. Kaplan, eds., Vol. 3, 891. Academic Press, New York, (1957). 8. DAVISSON, E. O., GIBSON, D. M., RAY, B. R., AND VESTLING, C. S., J. Phys. Chem. 57, 609 (1953). 9. FISHER, H.
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Chem. 237, 215 (1962). 12. GUTFREUND, H., CANTWELL, R.,
30. MCMLJRRAY,
COMPLEXES
575
C. H. CRIDDLE, R. S., AND HATHAWAY, G., Biochem. J., 106,683 (1968). HAKALA, M. T., GLAID, A. J., AND SCHWERT, G. W., J. Biol. Chem. 221, 191 (1956). HSIEH, W. T., AND VESTLING, C. S., Biochem. Prepamtions 11, 69 (1966). KAPLAN, N. O., CIOTTI, M. M., AND STOLZENBACH, F. E., Arch. Biochem. Biophys. 69, 441(1957). NYGAARD, A. P., AND THEORELL, H., Acta Chem. Scand. 9, 1300 (1955). OSER, B. L., Editor, Hawk’s Physiological Chemistry McGraw-Hill, New York, 14th Edition, p. 1243 (1965). PALIT, S. R., Ind. Eng. Chem., Anal. Ed. 18, 246 (1946). PFLEIDERER, G., AND HOHNHOLZ, E., Biochem. Z. 331, 245 (1959). RAVAL, D. N., AND WOLFE, R. G., Biochemistry 1, 1112 (1962). STEGINK, L. D., AND VESTLING, C. S., J. Biol. Chem. 241,4923 (1966). THEORELL, H., AND BONNICHSEN, R., Acta Chem. &and. 5, 1105 (1951). THEORELL, H., NYGAARD, A. P., AND BONNICHSEN, R., Acta Chem. &and. 9, 1148 (1955). THEORELL, H., AND WINER, A. D., Arch. Biothem. Biophys. 83,291(1959). THEOFELL, H., AND YONETANI, T., Biochem. z. 338, 537 (1963). THEORELL, H., AND YONETANI, T., Arch. Biothem. Biophys. 106, 252 (1964). VESTLING, C. S. in D. Glick, Editor, “Methods of Biochemical Analysis,” Interscience Publishers, Inc., New York, Vol. X, p. 137 (1962). WINER, A. D., Acta Chem. Scared. 17, Suppl. 1, 203( 1963). WRAT~EN, C. C., AND CLELAND, W. W., Biochemistry 2, 935 (1963). ZEWE, V., AND FROMM. H. J., J. Biol. Chem. 237, 1666 (1962).