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Fluorescence quenching of horse liver alcohol dehydrogenase and its complexes by para-chloromercuriphenyl sulfonate
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Fluorescence Dehydrogenase
166,
8-15 (197.5)
Quenching
of Horse
and Its Complexes by para-Chloromercuriphenyl Sulfonate l KAZUHIKO
Nobel Medical Institute,
Liver Alcohol
Department
TATEMOTO
Biochemistry, Enzyme Research Laboratory, Karolinska S-104 01 Stockholm, Sweden
of
Received
March
Institute,
13, 1974
The protein fluorescence quenching of horse liver alcohol dehydrogenase (LADH) by p-chloromercuriphenyl sulfonate (PMS) was studied in the presence and absence of various enzyme ligands. The protein fluorescence was quenched by PMS upon formation of the mercaptides with sulthydryl groups in LADH. The quenching of bound NADH fluorescence of the LADH-NADH and LADH-NADH-iso-butyramide complexes by PMS was studied comparatively and found to afford a method for investigating the mechanism of protein fluorescence quenching. The quenching mechanism appears to involve excitation energy transfer and denaturation of the enzyme. In addition, the release of ligand may also be involved in the mechanism. The formation of six enzyme-active modified forms of LADH by treatment of the enzyme with PMS was demonstrated by the fluorescence, activity, and electrophoretic measurements.
Protein fluorescence quenching techniques have not generally been used for investigation of the interaction between proteins and metal ions, in spite of many studies on the binding of metal ions to proteins. Lehrer (1) studied the quenching of protein fluorescence of transferrin by Fe3+ and Cu2+. Chen (2, 3) investigated systematically the fluorescence quenching of various proteins by mercuric and silver ions and suggested that the mechanism of quenching may involve complex formation with chromophores, excitation energy transfer, and conformational changes. It is known that the protein fluorescence of LADH2 is quenched on binding with
NADH, NAD+, or o-phenanthroline (4) and also by acid (5) or by urea (6). Although few data have been presented, the quenching of protein fluorescence of LADH by mercuric ions was noted by Chen (2). The interaction between LADH and p-chloromercuriphenyl sulfonate (PMS) appears suitable for studying the quenching mechanism, since PMS stoichiometrically binds to all 28 sulfhydryl groups in LADH molecule (7). NADH and NAD+ form the binary complexes LADH-NADH and LADH-NAD+ respectively at the active site of LADH (8). The fluorescence of NADH is increased approximately tenfold on binding with LADH when excited at 330 nm and emitted at 410 nm (9). Iso-butyramide competes with aldehyde to form the ternary complex LADH-NADH-iso-butyramide at the active site of LADH (10). The fluorescence of bound NADH in the ternary complex is further enchanced in the presence of iso-butyramide. Pyrazole inhibits LADH activity competitively with respect
‘This work was supported by grants from the Swedish Medical Research Council and from National Institute of Mental Health on Alcohol Abuse and Alcoholism (1ROl AAOO323-01). * The following abbreviations are used: LADH, E, horse liver alcohol dehydrogenase; NADH, reduced nicotinamide-adenine dinucleotide; NAD+, oxidized nicotinamide-adenine dinucleotide; PMS, pchloromercuriphenyl sulfonate.
8 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
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to alcohol and forms the ternary complex LADH-NAD+.-pyrazole at the active site (11). It has been reported that the characteristic fluorescence of bound NADH in the LADH-NADH and LADH-NADH-isobutyramide complexes is quenched by PMS (7). It is, therefore, of interest to study the quenching of the two different fluorescences comparatively. The data presented describe the effect of PMS on the protein fluorescence of LADH in the presence and absence of ligands in order to elucidate the structual and functional roles of the sulfhydryl groups in LADH. MATE:RIALS
AND
METHODS
Horse liver alcohol dehydrogenase (EE isoenzyme) was prepared according to Dalziel (12). An electrophoretically homogeneous preparation was obtained by chromatography on carboxymethyl cellulose (13) and recrystallization from 0.01 M phosphate buffer (pH 7.0) containing 8% ethanol. The crystals were stored in 35% ethanol at ~ 18°C. The enzyme solution was prepared by dissolving the crystals in phosphate-ammonium buffer (pH 9.3) and dialyzing against phosphate buffer (pH 7.0) for 3 days at 0°C. The enzyme concentration was determined by fluorimetric titration with NADH in the presence of 0.1 M iso-butyramide (10). Enzyme concentrations are expressed in micromolarity (WM) throughout this paper. Most chemicals were obtained from Sigma Chemical Co., and used without further purification; the exceptions were as follows: o-phenanthroline, Eastman Organic Chemicals; iso-butyramide, E. Merck AG., and pyrazole, Schuchardt, Miinchen. Assays of NADH and NAD+ were carried out on the basis of absorption indexes of 6.22 rnM-’ cm-’ at 340 nm and 18.0 rnM-’ cm-’ at 260 nm, respectively. The concentration of PMS was determined from the absorbance at 265 nm using an absorption index of 0.60 mM-’ cm-’ (7). The sulfhydryl content of LADH was determined spectrophotometrically using the method of Boyer (14), and fluorimetrically according to Yonetani and Theorell (7). Fluorimetric experiments were carried out using a recording spectrophotofluorimeter as described previously (4). The protein fluorescence of LADH was measured with excitation at :!97 nm and the bound NADH fluorescence was measured with excitation at 330 nm in all experiments. Enzyme activity measurements were carried out according to Dalziel (15). Initial rates were determined using OD 0.1 scale of a Cary 14A spectrophotometer. Electrophoresis on the cellulose acetate was performed in Microcell (Beckman) at 250 V for 1.5 hr
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in Tris buffer, pH 8.5 (I = 0.05). After electrophoresis, the enzyme-activity staining and protein staining were carried out as described by Pietruszko and Theorell (16). Experiments described were carried out at 23.5”C. RESULTS
The protein fluorescence quenching of free LADH by PMS. The protein fluorescence of free LADH was shown to be quenched by the addition of PMS. At pH 7.0 the stoichiometric addition of PMS to sulfhydryl groups of LADH caused a rapid quenching of the fluorescence excited at wavelength 297 nm (Fig. 1). PMS binds rapidly and specifically to the sulfhydryl groups to form mercaptides, this is probably the cause of the initial rapid quenching effect since PMS exhibits little light absorbance at these wavelengths. A small but significant increase in fluorescence, which was observed after this rapid decline, could be explained by the formation of LADH aggregates. The protein fluorescence quencing of LADH complexes by PMS. Since ligands in LADH complexes may give rise to quenching, the absolute quenching effect due to PMS may be difficult to interpret. It is well established that the protein fluorescence of LADH is quenched on binding with NADH, NAD+, or o-phenanthroline (4). The LADH fluorescence in the presence of saturated amount of NADH
WAVELENGTH
(nm)
FIG. 1. The effect of PMS on the fluorescence spectrum of LADH. The emission spectrum of native LADH (1.88 wM) (l), LADH (1.88 FM) 2 min (2) and 6 min (3) after the addition of 52.6 pM PMS in phosphate buffer (pH 7). The excitation wavelength was 297 nm.
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KAZUHIKA
was, therefore, approximately 36% of the free LADH under identical conditions and in the presence of NAD+ was 85%. After the addition of PMS to these complexes the fluorescence intensity in all cases was reduced to approximately 15%, similar to that for LADH bound to PMS; therefore indicating the probable release of the ligands from the complexes during the reaction. The fluorescence changes observed after the addition of PMS at different concentrations of NADH added to LADH are shown in Fig. 2. After the addition of PMS there was an immediate increase in the fluorescence which could represent the release of ligand. The magnitude of this increase was found to decrease with increasing concentration of ligand. After reaching a maximum the intensity of fluorescence was seen to decrease with time, and the final fluorescence intensity was similar in all cases and identical to that for LADH bound to PMS. Similar observations were found with the LADH-NAD+ and LADH-o-phenanthroline complexes, while no such findings were seen with the complexes LADH-AMP and LADH-ADPR. The effect of pyrazole on the protein fluorescence quenching of LADH-NAD+
TIME
TATEMOTO
by PMS is shown in Fig. 3. With increasing concentration of pyrazole, the degree of.the fluorescence quenching was diminished, indicating the protective effect of pyrazole against PMS by forming the LADH-NAD+ pyrazole complex (11). Comparative studies on the quenching of the protein and NADH fluorescence. The rate of change in the fluorescence of both protein and bound NADH of LADHNADH and LADH-NADH-iso-butyramide at pH 7.0 after the addition of PMS is islustrated in Fig. 4. A rapid disappearance in the bound NADH fluorescence of the LADH-NADH complex by PMS occurs corresponding to the rapid initial increase in the protein fluorescence of the complex (Fig. 2), thus, indicating that both fluorescence changes are probably due to the release of ligand. A slight difference between the quenching rate of the protein fluorescence and bound NADH fluorescence of LADHNADH-iso-butyramide was observed at pH 7. The difference was greater at pH 10 where 55% of the protein fluorescence of the complex was quenched rapidly by PMS, while little change was observed in the corresponding bound NADH fluorescence over the same period (Fig. 5). The rapid quenching at pH 10 was also observed for free LADH and the complexes of
-
FIG. 2. Changes in the fluorescence of LADH in the presence of NADH after the addition of PMS. The protein fluorescence was measured at 342 nm of LADH (0.24 pM) in the presence of 0, 0.83, 1.66, 4.15, and 8.3 C(M (curves 1-5, respectively) NADH in phosphate buffer (pH 7.0) after the addition of 20.8 )dM PMS. The arrows indicate the time of addition of PMS (close-tipped), and the time of the NADH additions (open-tipped). The excitation wavelength was 297 nm.
FIG. 3. The effect of pyrazole on the quenching of protein fluorescence of LADH-NAD+ complex by PMS. Curves l-8 show the changes in protein fluoresence of LADH (0.24 JLM) in the presence of NAD+ (1.14 mM) and pyrazole 0, 0.5, 1.0, 2.0, 10, 20, 100, and 670 pM, respectively, in phosphate buffer (pH 7.0) after the addition of PMS (12.5 PM). The excitation and emission wavelengths were 297 nm and 342 nm, respectively.
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;5 20\ 0, ‘*--..--.-.‘o--is ----3t-
30
TIME
( Set
40
TOW
TIME
--SC
(Sex)
FIG. 5. The fluorescence quenching at pH 10 of LADH complexes by PMS. Changes in the fluorescence were measured after the addition of PMS (42 pM) to (I) free LADH (0.24 fiM), (II) LADH-NADH complex (0.24 j1M:20 PM), (III) LADH-NADH-kobutyramide complex (0.24 /lM:1.6 ~~:0.1 M), in carbonate buffer pH 10 (I = 0.1). The protein fluorescence (excitation at 297 nm and emission at 342 nm) of (I) (O-O). (II) (H-B), and (III) (A-A) is indicated in addition to the NADH fluorescence (excitation at 330 nm and emission at 410 nm) of (II) (O-XI) and (III) (A-A).
LADH-NADH, LADH-NAD+, and LADH-NAD+-pyrazole and the rate of quenching was little affected by the presence of these ligands. To determine the number of reactive
11
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5
)
FIG. 4. The fluorescence quenching at pH 7.0 of LADH complexes, by PMS. PMS (12.5 NM) was added to free LADH (0.24 PM), the LADH-NADH complex (0.24 wM:4.4 PM], and the LDH-NADH-iso-butyramide complex (0.24 1M:d.J p~:O.l M) in phosphate buffer (pH 7.0), and changes in the protein fluorescence at 342 nm of free LADH (0-O) and LADH-NADH-iso-butyramide(A-A) and the bound NADH fluorescence at 410 nm of LADH-NADH (0-O) and LADH-NADH-iso-butyramide (A-A) were followed by a recording spectrophotofluorimeter. The excitation wavelengths of the protein and NADH fluorescences were 297 nm and 330 nm, respectively. To enable comparison, the changes in fluorescence are expressed as a percentage of the original fluorescence.
; PO! 4 ,” ---w
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15
10
[PMS]
20
OJM)
FIG. 6. The titration curves of LADH and its complexes with PMS at pH 10. LADH and the LADH complexes were titrated with PMS in carbonate buffer at pH 10 (I = 0.1). The quenching of protein fluorescence of free LADH (0.88 PM, O-O): LADHNADH complex (0.88 ~~:2.8 ,ttM, A-A): LADHNADH-iso-butyramide complex (0.88 r~:2.8 p~:O.l M, A-A) and LADH-NADH+-pyrazole complex (0.88 p~:7.6 FM, 0-O) was measured at 342 nm (excitation at 297 nm) after addition of PMS.
r~:670
groups involved in the quenching at pH 10, LADH and the complexes were titrated against PMS (Fig. 6). A linear decrease in the fluorescence was observed over the range O-5 pmoles of PMS/bmole of LADH in all cases, while at concentrations above 5 pmoles of PMS/hmole LADH, little or no change in the fluorescence of the complexes was observed. In contrast, after the addition of 5-6 pmoles of PMSlpmole LADH to LADH-NADH and LADH-NADH-isobutyramide, no change in the bound NADH fluorescence was observed. By the addition of reduced glutathione or dithioerythritol the protein fluorescence of these complexes was recovered. The total number of titratable groups with PMS at pH 10 was also measured using both the spectrophotometric method of Boyer (14) and the fluorimetric technique of Yonetani and Theorell (71, and found to be 28 per LADH molecule, indicating that PMS binds specifically to the sulfhydryl groups at pH 10. The rapid quenching at pH 10 is, therefore, due to interaction between PMS and the sulfhydryl groups and not between PMS and any other reactive groups in LADH. The fluorescence spectra at pH 10 of LADH and two of its ternary complexes before and after the addition of PMS are shown in Fig. 7. After the addition of PMS
12
KAZUHIKA
WAVE
LENGTH
(nm)
FIG. 7. The fluorescence spectra of LADH and its complexes at pH 10. The fluorescence spectra (excited at 297 nm) of LADH and its complexes in carbonate buffer (pH 10, I = 0.1) are illustrated for free LADH LADH-NAD+-pyrazole (0.9 ~~:7.6 (0.9 PM, -); __--. )i and LADH-NADH-ko-butyraj1~:670 PM, mide (0.9 p~:2.8 p~:O.l M, ---). The bold line indicates the spectra obtained before the addition of 4.15 MM PM.3 while the faint line corresponds to the respective spectra after the addition of PMS.
the protein fluorescence of LADH and its complexes was rapidly quenched with the fluorescence peak shifting to a lower wavelength. The fluorescence peaks at 435 nm of bound NADH are due to the excitation of NADH molecule in the complex through the protein excitation. After the addition of PMS to the LADH-NADH-iso-butyramide complex the fluorescence at 435 nm was partly quenched with no appreciable shift in the wavelength. It is, therefore, indicated that the formation of the melcaptides in the enzyme molecule quenched the protein excitation. Modification of LADH actiuity by PMS. At pH 10 a loss of 50-60s of the LADH activity was observed after the addition of 6 pmoles of PMS/pmole of free LADH or LADH-NADH-iso-butyramide (Fig. 8); however, after the addition of dithioerythritol the enzyme activity was restored. Preliminary kinetic experiments with a LADH preparation treated with 6 pmoles of PMS/pmole of enzyme indicated that the values for both the V and K, ([ethanol], [acetaldehyde] = co) of the modified LADH were approximately 50% that of the native LADH. Formation of the modified LADH was also observed by electrophoresis, since each mercaptide formed in the enzyme
TATEMOTO
adds one extra negative charge. The electrophoretic patterns of LADH before and after treatment with different amounts of PMS are illustrated in Fig. 9. After activity staining, a total of six distinctive bands were visible, and the composition of the electrophoretic pattern was seen to vary according to the PMWLADH concentration ratio. Addition of more than 6 pmoles PMS/ pmole LADH resulted in the spread of bands far below the origin. Since activity staining failed to reveal these bands, they probably corresponded to denatured LADH. After the addition of dithioerythrito1 the modified active LADH was reconverted to native LADH; however, no effect was observed with the denatured LADH, as evidenced after electrophoresis. The mobilities of the electrophoretic bands in the native isoenzymes of LADH (E, E’, and E”) were found to be identical to those of the PMS-modified LADH (Fig. 9) and might be consistent with a difference of one negative charge between each isoenzyme. The spectrophotometric titration of LADH with PMS. The results of the titration of LADH with PMS at pH 10 using the mercaptide absorption band are illustrated
FIG. 8. The effect of PMS on the enzyme activity of free LADH (O-O) and LADH-NADH-iso-butyramide complex (A-A) at pH 10. The enzyme activity was determined after titration of free LADH (5 pM) and LADH-NADH-iso-butyramide (5 pM:18 p~:O.l M) with PMS at pH 10 (I = 0.1) in carbonate buffer. The reaction mixture of the complex was diluted 1:600 during the activity determinations in order to dissociate the complex and a correction for the inhibition due to the presence of ko-butylamide was applied. The abscissa is expressed as molar ratio between PMS and LADH.
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FIG. 9. The electrophoresis of LADH in the presence of PMS at pH 10. The electrophoresis on cellulose acetate of (1) a mixture of LADH (E) and its native isoenzymes E’, E”: (2) homogeneous LADH and (3-8) LADH after the addition of 1.5, 3.0, 4.5, 6.0, 13.5, and 21.0 PM of PMS/FM LADH, respectively.
in Fig. 10. At 255 nm, a linear plot resulted, while at the maxium absorption increase (235 nm) the titration curve consisted of two linear lines, one over the range O-6 pmoles PMS/pmole LADH and the other over the range 6-28 pmoles PMS/pmole LADH. At pH 7, however, the titration curves showed a linear increase over the range O-28 Almoles PMS/gmole LADH at 235 nm. Quenching of protein fluorescence of LADH by the other heavy metal compounds. The protein fluorescence of LADH was found to be quenched by addition of HgC12, AgNO,, or CuSO,. At pH 7, the titration of LADH with PMS using the protein fluorescence quenching indicated the presence of 24 i 2 sulfhydryl groups, while with AgNO, this value was found to be 28. It was difficult to reproduce titration
results of PMS or HgCl, at pH 7 probably due to the formation of aggregates.
[PM’S]
(liM1
FIG. 10. The spectrophotometric titration of LADH with PMS at pH 10. LADH (4 PM) in carbonate buffer (pH 10, I = 0.1) was titrated with PMS and changes in light absorbance at 235 nm (O-O) and 255 nm (O-O) were measured. The absorption of PMS was automatically subtracted using a pair of reference cuvettes.
14
KAZUHIKA DISCUSSION
The complexes LADH-NADH and LADH-NADH-iso-butyramide exhibit both protein fluorescence and a characteristic fluorescence of bound NADH, the intensities of the latter being higher than that of free NADH (9, 10). When PMS is added to these complexes at pH 7, the bound NADH fluorescence is quenched to a level corresponding to that of free NADH. At pH 10, however, it remains unchanged by addition of 6 pmole PMS/pmole LADH complex, while the protein fluorescence of these complexes is quenched rapidly by the same addition. The absence of any change in the bound NADH fluorescence indicates that the protein structure and function is well preserved; therefore, suggesting that denaturation of the enzyme is not the cause of this rapid protein fluorescence quenching. The quenching effect of PMS is diminished upon addition of a reducing reagent such as reduced glutathione or dithioerythritol. These results, together with activity and electrophoretic measurements, indicate that the introduction of PMS to the LADH molecule at, pH 10 gives rise to modified forms of LADH which are still active; and that excitation energy transfer may occur from the tryptophan residues of LADH to the tail end of the mercaptide absorption band, as it was suggested by Chen (2). Kinetic experiments indicate that the presence of mercaptides in modified forms of LADH influence the rate of coenzyme dissociation from the apoenzyme but do not affect the rates of association of coenzyme and substrate. By the addition of more than 6 pmoles of PMS per pmole of LADH-NADH or LADH-NADH-iso-butyramide complex at pH 10, a slow quenching of the bound NADH fluorescence was observed. At pH 10, therefore, PMS binds initially to six sulfhydryl groups of the LADH and forms the modified LADH which retains its enzyme activity and binding to the remaining sulfhydryl groups results in denaturation of the enzyme. At pH 7, however, PMS binds to all sulfhydryl groups at almost identical rates and, therefore, only the denatured
- I‘Kl’J3MO’I’O --^-
enzyme is formed. Although the pH effect on the reactivity of PMS toward sulfhydryl group of LADH is difficult to interpret, it is suggested that the conformation of LADH at pH 10 might be different from that at pH 7 so that there are six sulfhydryl groups in the LADH molecule which are more susceptible to PMS. The results above indicate that the protein fluorescence quenching of LADH by PMS may involve at least two mechanism; (1) excitation energy transfer from the aromatic amino acid residues of LADH to the mercaptides formed on reaction with PMS, (2) conformational changes due to denaturation of the enzyme by the chemical binding. The possible complex formation between PMS and the aromatic amino acid residues in the molecule can be ruled out since PMS will bind only to the sulfhydryl groups of LADH. The protein fluorescence quenching by PMS in the presence of ligands may involve additional mechanisms since the ligands may also give rise to quenching. The release of NADH from the LADH-NADH complex on reaction with PMS resulted in an initial rapid recovery in the fluorescence. AMP and ADPR, which do not quench the fluorescence of LADH on binding, therefore, have little effect on the fluorescence quenching by PMS. Lutstorf et al. (17, ~18) have suggested that the native isoenzymes of LADH observed in electrophoresis of a LADH preparation (excluding the “steroid active” isoenzymes) arise by differences in conformation, although there is no positive evidence for this. A comparison between the electrophoretic data of the PMS-modified LADH and the native isoenzymes appear to indicate a difference of one negative charge between each isoenzyme. It is, therefore, possible that these charge differences account for the difference in the electrophoretie mobilities of the isoenzymes. ACKNOWLEDGMENTS The author thanks Professor Hugo Theorell for his interesting and helpful discussions. Thanks are also due to Dr. K. D. R. Setchell for help in the preparation of the manuscript.
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REFERENCES 1. LEHRER, S. S. (1961) J. Viol. Chem. 244, 3613-3617. 2. CHEN, R. F. (1971) Arch. Biochem. Biophys 142, 552-564. 3. CHEN, R. F. (1973) Arch. Biochem. Biophys. 158, 605-622. 4. THEORELL, H., AND TATEMOTO, K. (1971) Arch. Biochem. Biophys. 142, 69-82. 5. BLOMQUIST, C. H. (1967) Arch. Biochem. Biophys. 122, 24-31. 6. BRNAD, L., E~ERSE, J., AND KAPLAN, N. 0. (1962) Biochemistry 1, 423-434. 7. YONETANI, T., AND THEORELL, H. (1962) Arch. Biochem. Biophys. 99, 433-446. 8. THEORELL, H., AND BONNICHSEN, R. (1951) Acta Chem. Scarzd. 5, 1105-1126. 9. THEORELL, H., AND WINER, A. D. (1959) Arch. Biochem. Biophys. 83, 291-308.
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10. WINER, A. D., AND THEORELL, H. (1960) Acta Chem. &and. 14, 1729-1742. 11. THEORELL, H., AND YONETANI, T. (1963) Biochem. 2. 338, 537-553. 12. DALZIEL, K. (1958) Acta Chem. Stand. 12, 459-464. 13. TANICWCHI, S., THEORELL, H., AND AKESON, A. (1967) Acta Chem. Stand. 21, 1903-1920. 14. BOYER, P. D. (1954) J. Amer. them. Sot. 76, 4331-4337. 15. DALZIEL, K. (1957) Acta Chem. Stand. 11, 397-398. 16. PIETRUSZKO, R., AND THEORELL, H. (1969) Arch. Biochem. Biophys. 131, 288-298. 17. LUTSTORF, U. M., AND VON WARTBURG, J. P. (1969) Fed. Eur. Biochem. Sot. Lett. 5, 202-206. 18. LUTSTORF, U. M., SCHURCH, P. M., AND VON WARTBURG, J. P. (1970) Eur. J. Biochem. 17, 497-508.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Fluorescence Dehydrogenase
166,
8-15 (197.5)
Quenching
of Horse
and Its Complexes by para-Chloromercuriphenyl Sulfonate l KAZUHIKO
Nobel Medical Institute,
Liver Alcohol
Department
TATEMOTO
Biochemistry, Enzyme Research Laboratory, Karolinska S-104 01 Stockholm, Sweden
of
Received
March
Institute,
13, 1974
The protein fluorescence quenching of horse liver alcohol dehydrogenase (LADH) by p-chloromercuriphenyl sulfonate (PMS) was studied in the presence and absence of various enzyme ligands. The protein fluorescence was quenched by PMS upon formation of the mercaptides with sulthydryl groups in LADH. The quenching of bound NADH fluorescence of the LADH-NADH and LADH-NADH-iso-butyramide complexes by PMS was studied comparatively and found to afford a method for investigating the mechanism of protein fluorescence quenching. The quenching mechanism appears to involve excitation energy transfer and denaturation of the enzyme. In addition, the release of ligand may also be involved in the mechanism. The formation of six enzyme-active modified forms of LADH by treatment of the enzyme with PMS was demonstrated by the fluorescence, activity, and electrophoretic measurements.
Protein fluorescence quenching techniques have not generally been used for investigation of the interaction between proteins and metal ions, in spite of many studies on the binding of metal ions to proteins. Lehrer (1) studied the quenching of protein fluorescence of transferrin by Fe3+ and Cu2+. Chen (2, 3) investigated systematically the fluorescence quenching of various proteins by mercuric and silver ions and suggested that the mechanism of quenching may involve complex formation with chromophores, excitation energy transfer, and conformational changes. It is known that the protein fluorescence of LADH2 is quenched on binding with
NADH, NAD+, or o-phenanthroline (4) and also by acid (5) or by urea (6). Although few data have been presented, the quenching of protein fluorescence of LADH by mercuric ions was noted by Chen (2). The interaction between LADH and p-chloromercuriphenyl sulfonate (PMS) appears suitable for studying the quenching mechanism, since PMS stoichiometrically binds to all 28 sulfhydryl groups in LADH molecule (7). NADH and NAD+ form the binary complexes LADH-NADH and LADH-NAD+ respectively at the active site of LADH (8). The fluorescence of NADH is increased approximately tenfold on binding with LADH when excited at 330 nm and emitted at 410 nm (9). Iso-butyramide competes with aldehyde to form the ternary complex LADH-NADH-iso-butyramide at the active site of LADH (10). The fluorescence of bound NADH in the ternary complex is further enchanced in the presence of iso-butyramide. Pyrazole inhibits LADH activity competitively with respect
‘This work was supported by grants from the Swedish Medical Research Council and from National Institute of Mental Health on Alcohol Abuse and Alcoholism (1ROl AAOO323-01). * The following abbreviations are used: LADH, E, horse liver alcohol dehydrogenase; NADH, reduced nicotinamide-adenine dinucleotide; NAD+, oxidized nicotinamide-adenine dinucleotide; PMS, pchloromercuriphenyl sulfonate.
8 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
FLUORESCENCE
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to alcohol and forms the ternary complex LADH-NAD+.-pyrazole at the active site (11). It has been reported that the characteristic fluorescence of bound NADH in the LADH-NADH and LADH-NADH-isobutyramide complexes is quenched by PMS (7). It is, therefore, of interest to study the quenching of the two different fluorescences comparatively. The data presented describe the effect of PMS on the protein fluorescence of LADH in the presence and absence of ligands in order to elucidate the structual and functional roles of the sulfhydryl groups in LADH. MATE:RIALS
AND
METHODS
Horse liver alcohol dehydrogenase (EE isoenzyme) was prepared according to Dalziel (12). An electrophoretically homogeneous preparation was obtained by chromatography on carboxymethyl cellulose (13) and recrystallization from 0.01 M phosphate buffer (pH 7.0) containing 8% ethanol. The crystals were stored in 35% ethanol at ~ 18°C. The enzyme solution was prepared by dissolving the crystals in phosphate-ammonium buffer (pH 9.3) and dialyzing against phosphate buffer (pH 7.0) for 3 days at 0°C. The enzyme concentration was determined by fluorimetric titration with NADH in the presence of 0.1 M iso-butyramide (10). Enzyme concentrations are expressed in micromolarity (WM) throughout this paper. Most chemicals were obtained from Sigma Chemical Co., and used without further purification; the exceptions were as follows: o-phenanthroline, Eastman Organic Chemicals; iso-butyramide, E. Merck AG., and pyrazole, Schuchardt, Miinchen. Assays of NADH and NAD+ were carried out on the basis of absorption indexes of 6.22 rnM-’ cm-’ at 340 nm and 18.0 rnM-’ cm-’ at 260 nm, respectively. The concentration of PMS was determined from the absorbance at 265 nm using an absorption index of 0.60 mM-’ cm-’ (7). The sulfhydryl content of LADH was determined spectrophotometrically using the method of Boyer (14), and fluorimetrically according to Yonetani and Theorell (7). Fluorimetric experiments were carried out using a recording spectrophotofluorimeter as described previously (4). The protein fluorescence of LADH was measured with excitation at :!97 nm and the bound NADH fluorescence was measured with excitation at 330 nm in all experiments. Enzyme activity measurements were carried out according to Dalziel (15). Initial rates were determined using OD 0.1 scale of a Cary 14A spectrophotometer. Electrophoresis on the cellulose acetate was performed in Microcell (Beckman) at 250 V for 1.5 hr
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in Tris buffer, pH 8.5 (I = 0.05). After electrophoresis, the enzyme-activity staining and protein staining were carried out as described by Pietruszko and Theorell (16). Experiments described were carried out at 23.5”C. RESULTS
The protein fluorescence quenching of free LADH by PMS. The protein fluorescence of free LADH was shown to be quenched by the addition of PMS. At pH 7.0 the stoichiometric addition of PMS to sulfhydryl groups of LADH caused a rapid quenching of the fluorescence excited at wavelength 297 nm (Fig. 1). PMS binds rapidly and specifically to the sulfhydryl groups to form mercaptides, this is probably the cause of the initial rapid quenching effect since PMS exhibits little light absorbance at these wavelengths. A small but significant increase in fluorescence, which was observed after this rapid decline, could be explained by the formation of LADH aggregates. The protein fluorescence quencing of LADH complexes by PMS. Since ligands in LADH complexes may give rise to quenching, the absolute quenching effect due to PMS may be difficult to interpret. It is well established that the protein fluorescence of LADH is quenched on binding with NADH, NAD+, or o-phenanthroline (4). The LADH fluorescence in the presence of saturated amount of NADH
WAVELENGTH
(nm)
FIG. 1. The effect of PMS on the fluorescence spectrum of LADH. The emission spectrum of native LADH (1.88 wM) (l), LADH (1.88 FM) 2 min (2) and 6 min (3) after the addition of 52.6 pM PMS in phosphate buffer (pH 7). The excitation wavelength was 297 nm.
10
KAZUHIKA
was, therefore, approximately 36% of the free LADH under identical conditions and in the presence of NAD+ was 85%. After the addition of PMS to these complexes the fluorescence intensity in all cases was reduced to approximately 15%, similar to that for LADH bound to PMS; therefore indicating the probable release of the ligands from the complexes during the reaction. The fluorescence changes observed after the addition of PMS at different concentrations of NADH added to LADH are shown in Fig. 2. After the addition of PMS there was an immediate increase in the fluorescence which could represent the release of ligand. The magnitude of this increase was found to decrease with increasing concentration of ligand. After reaching a maximum the intensity of fluorescence was seen to decrease with time, and the final fluorescence intensity was similar in all cases and identical to that for LADH bound to PMS. Similar observations were found with the LADH-NAD+ and LADH-o-phenanthroline complexes, while no such findings were seen with the complexes LADH-AMP and LADH-ADPR. The effect of pyrazole on the protein fluorescence quenching of LADH-NAD+
TIME
TATEMOTO
by PMS is shown in Fig. 3. With increasing concentration of pyrazole, the degree of.the fluorescence quenching was diminished, indicating the protective effect of pyrazole against PMS by forming the LADH-NAD+ pyrazole complex (11). Comparative studies on the quenching of the protein and NADH fluorescence. The rate of change in the fluorescence of both protein and bound NADH of LADHNADH and LADH-NADH-iso-butyramide at pH 7.0 after the addition of PMS is islustrated in Fig. 4. A rapid disappearance in the bound NADH fluorescence of the LADH-NADH complex by PMS occurs corresponding to the rapid initial increase in the protein fluorescence of the complex (Fig. 2), thus, indicating that both fluorescence changes are probably due to the release of ligand. A slight difference between the quenching rate of the protein fluorescence and bound NADH fluorescence of LADHNADH-iso-butyramide was observed at pH 7. The difference was greater at pH 10 where 55% of the protein fluorescence of the complex was quenched rapidly by PMS, while little change was observed in the corresponding bound NADH fluorescence over the same period (Fig. 5). The rapid quenching at pH 10 was also observed for free LADH and the complexes of
-
FIG. 2. Changes in the fluorescence of LADH in the presence of NADH after the addition of PMS. The protein fluorescence was measured at 342 nm of LADH (0.24 pM) in the presence of 0, 0.83, 1.66, 4.15, and 8.3 C(M (curves 1-5, respectively) NADH in phosphate buffer (pH 7.0) after the addition of 20.8 )dM PMS. The arrows indicate the time of addition of PMS (close-tipped), and the time of the NADH additions (open-tipped). The excitation wavelength was 297 nm.
FIG. 3. The effect of pyrazole on the quenching of protein fluorescence of LADH-NAD+ complex by PMS. Curves l-8 show the changes in protein fluoresence of LADH (0.24 JLM) in the presence of NAD+ (1.14 mM) and pyrazole 0, 0.5, 1.0, 2.0, 10, 20, 100, and 670 pM, respectively, in phosphate buffer (pH 7.0) after the addition of PMS (12.5 PM). The excitation and emission wavelengths were 297 nm and 342 nm, respectively.
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;5 20\ 0, ‘*--..--.-.‘o--is ----3t-
30
TIME
( Set
40
TOW
TIME
--SC
(Sex)
FIG. 5. The fluorescence quenching at pH 10 of LADH complexes by PMS. Changes in the fluorescence were measured after the addition of PMS (42 pM) to (I) free LADH (0.24 fiM), (II) LADH-NADH complex (0.24 j1M:20 PM), (III) LADH-NADH-kobutyramide complex (0.24 /lM:1.6 ~~:0.1 M), in carbonate buffer pH 10 (I = 0.1). The protein fluorescence (excitation at 297 nm and emission at 342 nm) of (I) (O-O). (II) (H-B), and (III) (A-A) is indicated in addition to the NADH fluorescence (excitation at 330 nm and emission at 410 nm) of (II) (O-XI) and (III) (A-A).
LADH-NADH, LADH-NAD+, and LADH-NAD+-pyrazole and the rate of quenching was little affected by the presence of these ligands. To determine the number of reactive
11
DEHYDROGENASE
5
)
FIG. 4. The fluorescence quenching at pH 7.0 of LADH complexes, by PMS. PMS (12.5 NM) was added to free LADH (0.24 PM), the LADH-NADH complex (0.24 wM:4.4 PM], and the LDH-NADH-iso-butyramide complex (0.24 1M:d.J p~:O.l M) in phosphate buffer (pH 7.0), and changes in the protein fluorescence at 342 nm of free LADH (0-O) and LADH-NADH-iso-butyramide(A-A) and the bound NADH fluorescence at 410 nm of LADH-NADH (0-O) and LADH-NADH-iso-butyramide (A-A) were followed by a recording spectrophotofluorimeter. The excitation wavelengths of the protein and NADH fluorescences were 297 nm and 330 nm, respectively. To enable comparison, the changes in fluorescence are expressed as a percentage of the original fluorescence.
; PO! 4 ,” ---w
OF ALCOHOL
15
10
[PMS]
20
OJM)
FIG. 6. The titration curves of LADH and its complexes with PMS at pH 10. LADH and the LADH complexes were titrated with PMS in carbonate buffer at pH 10 (I = 0.1). The quenching of protein fluorescence of free LADH (0.88 PM, O-O): LADHNADH complex (0.88 ~~:2.8 ,ttM, A-A): LADHNADH-iso-butyramide complex (0.88 r~:2.8 p~:O.l M, A-A) and LADH-NADH+-pyrazole complex (0.88 p~:7.6 FM, 0-O) was measured at 342 nm (excitation at 297 nm) after addition of PMS.
r~:670
groups involved in the quenching at pH 10, LADH and the complexes were titrated against PMS (Fig. 6). A linear decrease in the fluorescence was observed over the range O-5 pmoles of PMS/bmole of LADH in all cases, while at concentrations above 5 pmoles of PMS/hmole LADH, little or no change in the fluorescence of the complexes was observed. In contrast, after the addition of 5-6 pmoles of PMSlpmole LADH to LADH-NADH and LADH-NADH-isobutyramide, no change in the bound NADH fluorescence was observed. By the addition of reduced glutathione or dithioerythritol the protein fluorescence of these complexes was recovered. The total number of titratable groups with PMS at pH 10 was also measured using both the spectrophotometric method of Boyer (14) and the fluorimetric technique of Yonetani and Theorell (71, and found to be 28 per LADH molecule, indicating that PMS binds specifically to the sulfhydryl groups at pH 10. The rapid quenching at pH 10 is, therefore, due to interaction between PMS and the sulfhydryl groups and not between PMS and any other reactive groups in LADH. The fluorescence spectra at pH 10 of LADH and two of its ternary complexes before and after the addition of PMS are shown in Fig. 7. After the addition of PMS
12
KAZUHIKA
WAVE
LENGTH
(nm)
FIG. 7. The fluorescence spectra of LADH and its complexes at pH 10. The fluorescence spectra (excited at 297 nm) of LADH and its complexes in carbonate buffer (pH 10, I = 0.1) are illustrated for free LADH LADH-NAD+-pyrazole (0.9 ~~:7.6 (0.9 PM, -); __--. )i and LADH-NADH-ko-butyraj1~:670 PM, mide (0.9 p~:2.8 p~:O.l M, ---). The bold line indicates the spectra obtained before the addition of 4.15 MM PM.3 while the faint line corresponds to the respective spectra after the addition of PMS.
the protein fluorescence of LADH and its complexes was rapidly quenched with the fluorescence peak shifting to a lower wavelength. The fluorescence peaks at 435 nm of bound NADH are due to the excitation of NADH molecule in the complex through the protein excitation. After the addition of PMS to the LADH-NADH-iso-butyramide complex the fluorescence at 435 nm was partly quenched with no appreciable shift in the wavelength. It is, therefore, indicated that the formation of the melcaptides in the enzyme molecule quenched the protein excitation. Modification of LADH actiuity by PMS. At pH 10 a loss of 50-60s of the LADH activity was observed after the addition of 6 pmoles of PMS/pmole of free LADH or LADH-NADH-iso-butyramide (Fig. 8); however, after the addition of dithioerythritol the enzyme activity was restored. Preliminary kinetic experiments with a LADH preparation treated with 6 pmoles of PMS/pmole of enzyme indicated that the values for both the V and K, ([ethanol], [acetaldehyde] = co) of the modified LADH were approximately 50% that of the native LADH. Formation of the modified LADH was also observed by electrophoresis, since each mercaptide formed in the enzyme
TATEMOTO
adds one extra negative charge. The electrophoretic patterns of LADH before and after treatment with different amounts of PMS are illustrated in Fig. 9. After activity staining, a total of six distinctive bands were visible, and the composition of the electrophoretic pattern was seen to vary according to the PMWLADH concentration ratio. Addition of more than 6 pmoles PMS/ pmole LADH resulted in the spread of bands far below the origin. Since activity staining failed to reveal these bands, they probably corresponded to denatured LADH. After the addition of dithioerythrito1 the modified active LADH was reconverted to native LADH; however, no effect was observed with the denatured LADH, as evidenced after electrophoresis. The mobilities of the electrophoretic bands in the native isoenzymes of LADH (E, E’, and E”) were found to be identical to those of the PMS-modified LADH (Fig. 9) and might be consistent with a difference of one negative charge between each isoenzyme. The spectrophotometric titration of LADH with PMS. The results of the titration of LADH with PMS at pH 10 using the mercaptide absorption band are illustrated
FIG. 8. The effect of PMS on the enzyme activity of free LADH (O-O) and LADH-NADH-iso-butyramide complex (A-A) at pH 10. The enzyme activity was determined after titration of free LADH (5 pM) and LADH-NADH-iso-butyramide (5 pM:18 p~:O.l M) with PMS at pH 10 (I = 0.1) in carbonate buffer. The reaction mixture of the complex was diluted 1:600 during the activity determinations in order to dissociate the complex and a correction for the inhibition due to the presence of ko-butylamide was applied. The abscissa is expressed as molar ratio between PMS and LADH.
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DEHYDROGENASE
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0+
FIG. 9. The electrophoresis of LADH in the presence of PMS at pH 10. The electrophoresis on cellulose acetate of (1) a mixture of LADH (E) and its native isoenzymes E’, E”: (2) homogeneous LADH and (3-8) LADH after the addition of 1.5, 3.0, 4.5, 6.0, 13.5, and 21.0 PM of PMS/FM LADH, respectively.
in Fig. 10. At 255 nm, a linear plot resulted, while at the maxium absorption increase (235 nm) the titration curve consisted of two linear lines, one over the range O-6 pmoles PMS/pmole LADH and the other over the range 6-28 pmoles PMS/pmole LADH. At pH 7, however, the titration curves showed a linear increase over the range O-28 Almoles PMS/gmole LADH at 235 nm. Quenching of protein fluorescence of LADH by the other heavy metal compounds. The protein fluorescence of LADH was found to be quenched by addition of HgC12, AgNO,, or CuSO,. At pH 7, the titration of LADH with PMS using the protein fluorescence quenching indicated the presence of 24 i 2 sulfhydryl groups, while with AgNO, this value was found to be 28. It was difficult to reproduce titration
results of PMS or HgCl, at pH 7 probably due to the formation of aggregates.
[PM’S]
(liM1
FIG. 10. The spectrophotometric titration of LADH with PMS at pH 10. LADH (4 PM) in carbonate buffer (pH 10, I = 0.1) was titrated with PMS and changes in light absorbance at 235 nm (O-O) and 255 nm (O-O) were measured. The absorption of PMS was automatically subtracted using a pair of reference cuvettes.
14
KAZUHIKA DISCUSSION
The complexes LADH-NADH and LADH-NADH-iso-butyramide exhibit both protein fluorescence and a characteristic fluorescence of bound NADH, the intensities of the latter being higher than that of free NADH (9, 10). When PMS is added to these complexes at pH 7, the bound NADH fluorescence is quenched to a level corresponding to that of free NADH. At pH 10, however, it remains unchanged by addition of 6 pmole PMS/pmole LADH complex, while the protein fluorescence of these complexes is quenched rapidly by the same addition. The absence of any change in the bound NADH fluorescence indicates that the protein structure and function is well preserved; therefore, suggesting that denaturation of the enzyme is not the cause of this rapid protein fluorescence quenching. The quenching effect of PMS is diminished upon addition of a reducing reagent such as reduced glutathione or dithioerythritol. These results, together with activity and electrophoretic measurements, indicate that the introduction of PMS to the LADH molecule at, pH 10 gives rise to modified forms of LADH which are still active; and that excitation energy transfer may occur from the tryptophan residues of LADH to the tail end of the mercaptide absorption band, as it was suggested by Chen (2). Kinetic experiments indicate that the presence of mercaptides in modified forms of LADH influence the rate of coenzyme dissociation from the apoenzyme but do not affect the rates of association of coenzyme and substrate. By the addition of more than 6 pmoles of PMS per pmole of LADH-NADH or LADH-NADH-iso-butyramide complex at pH 10, a slow quenching of the bound NADH fluorescence was observed. At pH 10, therefore, PMS binds initially to six sulfhydryl groups of the LADH and forms the modified LADH which retains its enzyme activity and binding to the remaining sulfhydryl groups results in denaturation of the enzyme. At pH 7, however, PMS binds to all sulfhydryl groups at almost identical rates and, therefore, only the denatured
- I‘Kl’J3MO’I’O --^-
enzyme is formed. Although the pH effect on the reactivity of PMS toward sulfhydryl group of LADH is difficult to interpret, it is suggested that the conformation of LADH at pH 10 might be different from that at pH 7 so that there are six sulfhydryl groups in the LADH molecule which are more susceptible to PMS. The results above indicate that the protein fluorescence quenching of LADH by PMS may involve at least two mechanism; (1) excitation energy transfer from the aromatic amino acid residues of LADH to the mercaptides formed on reaction with PMS, (2) conformational changes due to denaturation of the enzyme by the chemical binding. The possible complex formation between PMS and the aromatic amino acid residues in the molecule can be ruled out since PMS will bind only to the sulfhydryl groups of LADH. The protein fluorescence quenching by PMS in the presence of ligands may involve additional mechanisms since the ligands may also give rise to quenching. The release of NADH from the LADH-NADH complex on reaction with PMS resulted in an initial rapid recovery in the fluorescence. AMP and ADPR, which do not quench the fluorescence of LADH on binding, therefore, have little effect on the fluorescence quenching by PMS. Lutstorf et al. (17, ~18) have suggested that the native isoenzymes of LADH observed in electrophoresis of a LADH preparation (excluding the “steroid active” isoenzymes) arise by differences in conformation, although there is no positive evidence for this. A comparison between the electrophoretic data of the PMS-modified LADH and the native isoenzymes appear to indicate a difference of one negative charge between each isoenzyme. It is, therefore, possible that these charge differences account for the difference in the electrophoretie mobilities of the isoenzymes. ACKNOWLEDGMENTS The author thanks Professor Hugo Theorell for his interesting and helpful discussions. Thanks are also due to Dr. K. D. R. Setchell for help in the preparation of the manuscript.
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REFERENCES 1. LEHRER, S. S. (1961) J. Viol. Chem. 244, 3613-3617. 2. CHEN, R. F. (1971) Arch. Biochem. Biophys 142, 552-564. 3. CHEN, R. F. (1973) Arch. Biochem. Biophys. 158, 605-622. 4. THEORELL, H., AND TATEMOTO, K. (1971) Arch. Biochem. Biophys. 142, 69-82. 5. BLOMQUIST, C. H. (1967) Arch. Biochem. Biophys. 122, 24-31. 6. BRNAD, L., E~ERSE, J., AND KAPLAN, N. 0. (1962) Biochemistry 1, 423-434. 7. YONETANI, T., AND THEORELL, H. (1962) Arch. Biochem. Biophys. 99, 433-446. 8. THEORELL, H., AND BONNICHSEN, R. (1951) Acta Chem. Scarzd. 5, 1105-1126. 9. THEORELL, H., AND WINER, A. D. (1959) Arch. Biochem. Biophys. 83, 291-308.
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10. WINER, A. D., AND THEORELL, H. (1960) Acta Chem. &and. 14, 1729-1742. 11. THEORELL, H., AND YONETANI, T. (1963) Biochem. 2. 338, 537-553. 12. DALZIEL, K. (1958) Acta Chem. Stand. 12, 459-464. 13. TANICWCHI, S., THEORELL, H., AND AKESON, A. (1967) Acta Chem. Stand. 21, 1903-1920. 14. BOYER, P. D. (1954) J. Amer. them. Sot. 76, 4331-4337. 15. DALZIEL, K. (1957) Acta Chem. Stand. 11, 397-398. 16. PIETRUSZKO, R., AND THEORELL, H. (1969) Arch. Biochem. Biophys. 131, 288-298. 17. LUTSTORF, U. M., AND VON WARTBURG, J. P. (1969) Fed. Eur. Biochem. Sot. Lett. 5, 202-206. 18. LUTSTORF, U. M., SCHURCH, P. M., AND VON WARTBURG, J. P. (1970) Eur. J. Biochem. 17, 497-508.