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Levy, H. B. and Sober, H. A. (1960), Proc. SOC.Exptl. Biol. 103, 250. Lichter, E. A., and Dray, S. (1964), J. Immunol. 92, 91. Mancini, G,, Vaerman, J. P., Carbonara, A. D., and Heremmans, J. F. (1963), Proc. XI Colloq. Protein Biol. Fluids 11, 370.
Natvig, J. B., Kunkel, H. G., and Litwin, S. D. (1967), Cold Spring Harbor Symp. Quant. Biol. 32, 173. Oudin, J. (1956), Compt. Rend. 242, 2606. Oudin, J. (1960), J. Exptl. Med. 112, 107. Pernis, B., Chiappino, M. B., Kelus, A. S., and Gel], P. (1965), J . Exptl. Med. 122, 853. Petras, M. L. (1963), Proc. Natl. Acad. Sci. U. S . 50, 112.
Reisfeld, R. A., Dray, S., and Nisonoff, A. (1965), Immunochemistry 2, 155. Schwartz, M., and Myers, T. C. (1958), Anal. Chem. 30, 1150. Small, P. A., Reisfeld, R. A,, and Dray, S. (1965), J . Mol. Biol. 11, 713. Small, P. A., Reisfeld, R. A,, and Dray, S. (1966), J. Mol. Biol. 16, 328. Takahashi, Y . , Aoyama, I., Ito, F., and Yamamura, Y. (1967), Clin. Chim. Acta 18, 21. Uriel, J. (1963), Ann. N . Y. Acad. Sci. 103, 956. Uriel, J. (1964), in Immunoelectrophoretic Analysis, Grabar, P., and Burton, P., Ed., Elsevier, Amsterdam, p 50. Williams, D. E., and Reisfeld, R. A. (1964), Ann. N . Y . Acad. Sci. 121, 373.
Dehydrogenase-Reduced Coenzyme Difference Spectra, Their Resolution and Relationship to the Stereospecificity of Hydrogen Transfer* Harvey F. Fisher, Dismus L. Adija, and Dallas G. Cross
ABSTRACT : The binding of reduced diphosphopyridine nucleotide to any specific dehydrogenase causes changes in the ultraviolet absorption spectrum of the reduced coenzyme. The 340-mp regions of the resulting difference spectra can all be resolved into two simple operations on a reduced diphosphopyridine nucleotide spectrum; a shift of the band itself to a higher or lower wavelength without any change in shape and a uniform hyper- or hypochromicity of that peak. We find that A stereospecific dehydrogenases produce blue shifts while B stereospecific dehydrogenases produce red shifts (with the pos-
I
n previous studies we have shown that the shift in the ultraviolet absorption spectrum of DPNH produced by the binding of the reduced coenzyme to either liver alcohol dehydrogenase, liver L-lactate dehydrogenase, or mitochondrial malate dehydrogenase described by Theorell and Bonnichsen (1951), Chance and Neilands (1952), and Pfleiderer and Hohnholz (1959) is not a unique property of those three enzymes, but occurs in the binary complexes of other dehydrogenases (Fisher and Cross, 1966). In a more recent paper (Cross and Fisher, 1969), we have shown that changes in the conformation
* From the Laboratory of Molecular Biochemistry, Veterans Administration Hospital, Kansas City, Missouri 64128, and the Department of Biochemistry, University of Kansas School of Medicine, Kansas City, Kansas. Received April 7, 1969. This work was supported in part by Grant GM15188 from the National Institutes of Health and Grant GB-39 15 from the National Science Foundation. A preliminary report of a portion of this work appeared in the abstracts sf the 1968 meeting of the Federation of American Societies of Experimental Biology (Cross and Fisher, 1968).
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FISHER, ADIJA, A N D C R O S S
sible exception of mitochondrial malate dehydrogenase). The size of most of the shifts observed here is sufficiently large that the concentration of reduced diphosphopyridine nucleotide involved can be calculated. We have previously shown that changes in conformation of reduced diphosphopyridine nucleotide produce difference spectra resolvable into various combinations of the same components. Most of the shifts of the coenzyme spectrum in dehydrogenase complexes, however, are far too large to be accounted for by a simple opening or closing of the reduced diphosphopyridine nucleotide molecule.
of DPNH (or TPNH) in solution generate rather similar difference spectra, and that all such spectra can be resolved into combinations of simple shifts and hypochromicities of the 340-mp band of DPNH. While the difference spectra resulting from DPNH conformational changes differ from those due to dehydrogenase complexing, and while no two dehydrogenase binary complex difference spectra are themselves identical, all of these difference spectra can be resolved into different combinations and algebraic senses of the same two simple operations that sufficed for the resolution of conformation difference spectra. We present here a general survey of the difference spectra of dehydrogenase-reduced coenzyme binary complexes, the resolutions of the reduced nicotinamide absorption regions of those difference spectra into two simple and physically relevant operations, and consider the possible implications of these resolutions. The scope of this particular paper is limited to those things which are characteristic of dehydrogenase-reduced coenzyme difference spectra as a group-more detailed studies of
VOL.
8,
TABLE I:
NO.
11,
NOVEMBER
1969
Resolution Components of the 340-mp Region of Dehydrogenase-Coenzyme Difference Spectra.a Enzyme Stereospecificity
z
Ax (md Required for Resolution
HYPO- or Hyperchromicity Required for Resolution
Amount of Coenzyme Required for Resolution ( p
Dehydrogenase (mg/ml)f
PH
Coenzyme (
Glucose 6-phosphate ( = l>e Glutamate (1 .O) Glutamate (0.88) a-Glycerol phosphate (0.844) 20-fl-Hydroxysteroid ( = l)e Glyceraldehyde-3-PO4 (1.13) Malate (mitochondrial) (1.27)
8.0b 7.6. 7.6 7.6.
TPNH (9.9) TPNH (100) DPNH (69) DPNH (9.9)
B B B B
Red Red Red Red
37 =t4 34 2 31 i 2 31 f 2
-44 f 5 -30 7 -20 f 5 -39 f 8
1.8 i0.6 2.4 + 0.1 2.0 i 0.4 4.1 i 0 . 2
7.7 8.7.
DPNH (12.4) DPNH (134)
B B
Red 29 f 2 Red 11 i 2
-3 i4 -25 i 5
2.8 i0.5 2 . 6 i 0.5
7.1
DPNH (25.6)
?
Red 22 i 1
+12 i 3
8.6 f 1.0
Alcohol (liver) (1 .O) Alcohol (yeast) (0.98) Lactate (0,93) Isocitrate (0.86) Malate (supernatant) (0.85)
7.2 7.2 7.6. 7.4d 7.6
DPNH (10) DPNH (73) DPNH(16) TPNH (10) DPNH (42)
A A A A A
Blue 14 i 1 Blue 5 i 1 Blue 2 i 1 Blue 2 i 1 Blue 4 i 2
-35 -22 -2 -4 0
p ~ )
*
*
i2
i2 f4
i8
1 2
~ )
10 i 1 13 i 2 17 i 2 13 i 8 15 f 8
a The buffer used was 0.1 M potassium phosphate except: b 0.05 M Tris, 0.2 M potassium phosphate, and d 0.25 M Tris. e Estimated from the dilution of stock solutions purchased from Sigma Chemical Corp. Concentration of dehydrogenase was calculated from the mg/ml extinction coefficient at 280 mp as follows: glutamate, 0.97 (Olson and Anfinsen, 1952); a-glycerophosphate, 0.75 (Beisenherz et al., 1955); glyceraldehyde 3-phosphate, 2.1 (Krebs et al., 1953); mitochondrial malate, 0.305 (Harada and Wolfe, 1968); liver alcohol, 0.42 (Shore and Theorell, 1966); yeast alcohol, 1.25 (Hayes and Velick, 1954); lactate, 1.42 (Velick, 1958); isocitrate, 0.90 (Moyle, 1965); and supernatant malate, 0.90 (P-L Biochemicals, Inc.).
’
specific features of individual enzyme-coenzyme and enzymecoenzyme-substrate complexes and the relation of such features to the kinetics and mechanism of action of the enzyme will be the subject of later papers in this series. Materials and Methods The following enzyme preparations were purchased from Sigma Chemical Co. ; the descriptions of the properties and methods of preparation were supplied by Mr. Louis Berger of that company (personal communication). Bovine liver L-glutamate dehydrogenase, type I, a highly purified, crystalline preparation is prepared substantially as described by Olson and Anfinsen (1952). The lactate dehydrogenase and malate dehydrogenase contamination amounts to less than 0.2 % of the glutamate dehydrogenase activity. Glucose 6-phosphate dehydrogenase, type VI, is a partially purified preparation from baker’s yeast. Although its specific activity was somewhat low, it did have relatively low contaminating enzyme activities being substantially free of hexokinase and 6-phosphogluconic dehydrogenase. Purification was achieved primarily through the use of ammonium sulfate fractionation and column chromatography. Liver alcohol dehydrogenase is a crystallized preparation, from horse liver prepared substantially as described by Bonnichsen and Brink (1955). Isdcitrate dehydrogenase, type IV, is a highly purified preparation from pig heart prepared substantially as described by Siebert et al. (1957). contaminating enzymes such
as aconitase and DPN-specific isocitrate dehydrogenase are of a low level (less than 0.2% of the TPN-specific isocitrate dehydrogenase activity). Lactate dehydrogenase, type 111, is a highly purified, crystalline preparation from beef heart prepared substantially as described by Meister (1952). Its pyruvate kinase content is less than 0.03% of its lactate dehydrogenase activity. a-Glycerophosphate dehydrogenase is a highly purified, crystalline preparation from rabbit muscle. It has been purified substantially as described by Beisenherz et al. (1955). It is substantially free of aldolase, lactate dehydrogenase, pyruvate kinase, and glyceraldehyde phosphate dehydrogenase. 20P-Hydroxysteroid dehydrogenase, type 11, is a highly purified preparation from Streptomyces hydrogenans. It is substantially free of DPNH oxidase. Na2DPNH (grade 111, 98 %) and TPNH (type 11) were also products of the Sigma Chemical Corp. Yeast alcohol dehydrogenase was purchased from the Mann Chemical Corp. Mitochondrial L-malate dehydrogenase (batch 71), prepared from pig heart by the method of Thorne and Cooper (1963), was a product of Seravac Laboratories. Supernatant L-malate dehydrogenase (lot SM-3), prepared by the method of Thorne and Cooper, was a product of P-L Biochemicals, Inc. Yeast glyceraldehyde 3-phosphate dehydrogenase was a gift from Dr. Archie L. Murdock, University of Kansas School of Medicine. All dehydrogenases were prepared by dialyzing them against their respective bufers shown in Table 1. In addition, both yeast and liver alcohol dehydrogenases were pretreated with a
D ~ H Y U K O G ~ N A S ~ - K ~ D UC CU E~ N D Z Y M LU I F F L K ~ N LC S P L C I K A
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BIOCHEMISTRY
were made to the enzyme solution in the sample compartment and to a cuvet containing only buffer in the reference compartment. All other procedures have been previously described (Cross and Fisher, 1969). Results
A(md FIGURE 1 : Dehydrogenase-reduced coenzyme difference spectra (solid lines) and the sum of the components required for resolution of the 340-mp region (dotted lines). Conditions and concentrations of enzymes and coenzymes are described in the Materials and Methods section and in Table I. Enzyme abbreviations are as follows: GAPDH, yeast glyceraldehyde 3-phosphate; PHSDH, 2OP-hydroxysteroid; G-6-PDH, glucose 6-phosphate; GDH, glutamate; (uGPDH, a-glycerol phosphate; mMDH, mitochondrial malate; LADH, liver alcohol; YADH, yeast alcohol; LDH, lactate; sin MDH, supernatant malate; and ICDH, isocitrate dehydrogenases.
5 x 10-6 M solution of DPNH before dialysis and with Norit A followed by filtration. After dialysis all enzymes were filtered through a 0.45 p Millipore filter. Difference spectra were recorded on a Cary Model 14 spectrophotometer using the zero- to one-tenth-scale expansion. Cuvets of 1.000-cm path length were used in a tandem cell arrangement as described by Herskovits and Laskowski (1961). The sample and reference compartments were thermostated to 20 =t0.2’. Base lines were recorded with enzyme in both sample and reference compartments. Additions of coenzyme
4426
FISHER,
ADIJA,
AND
CROSS
The difference spectra of eleven dehydrogenase-reduced coenzyme binary complexes (as referred to separated components) are shown as solid lines in Figure 1. The dotted lines represent summations of resolved components to be described later. Such difference spectra for supernatant malate, glucose &phosphate, 20-/3-hydroxysteroid, and a-glycerolphosphate dehydrogenases have not been previously reported. Resolution of ER Difference Spectra. PROCEDURE. In a previous paper (Cross and Fisher, 1969), we pointed out that any resolution of a complex curve is not in itself unique and proposed several criteria to ensure that the components and operations chosen would correspond to physically meaningful entities. We also showed that the 340-mp region of all difference spectra generated by conformational changes of DPNH could be resolved into a shift in wavelength and a hypo- or hyperchromicityl of the original total spectrum. The use of these two operations was justified on the basis that a shift in wavelength is to be expected from any change in the intermediate environment of a chromophore; that the requirements for hypochromism as stated by Tinoco are present in the DPNH molecule and the phenomenon itself is common in polynucleotide systems; and that all difference spectra obtained could be successfully resolved without assuming that the 340-rn~ band contained more than a single electronic transition. While we will use only these same two operations to resolve the enzyme complex difference spectra, there is one important quantitative difference in the resolutions of the difference spectra reported previously and those described here. The difference spectra generated by conformational changes in the DPNH molecule could all be adequately resolved using only very small spectral shifts (
