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A pressure jump apparatus with optical detection
ANALYTICAL
BIOCHEMISTBY
A Pressure
28,
Jump
Research
Board
(1969)
Apparatus
D. E. GOLDSACK’, Fisheties
273-281
of
with
R. E. HURST, Canada, Vancouver B. C., Canada
Received August
Optical AND
Detection J. LOVE
Laboratory,
Vancouver
8,
9, 1968
The recent introduction of perturbation techniques into chemical kinetic studies in solution has now made possible studies of chemical reactions whose reaction times fall in the second to microsecond region (1). These techniques have already been used to investigate fundamental biophysical problems such as the transient behavior of enzyme reactions and the isomerization of proteins (2,3). The essence of these perturbation techniques is to take a chemical system in a state of equilibrium and to quickly perturb the system to a new equilibrium state by a sudden change in an external variable such as temperature, pressure, or electric field strength (1). If the change in the external variable is fast enough (1 to 50 psec) , then chemical reactions which are slower than this change can be followed by appropriate monitoring techniques. For studies of protein systems the two methods applied so far have been the temperature jump technique (2) and the pressure jump method (3). The temperature jump technique involves creating a fast temperature rise in a highly conductive aqueous solution by joule heating, and the chemical change is monitored optically by following the small change in absorbance of the system. The pressure jump technique on the other hand involves application of a static pressure to an aqueous solution; a sharp drop in pressure is produced by a bursting membrane technique. In this case the system is monitored by following the small changes in conductivity of the solution. The requirement of a highly conductive solution in the temperature jump technique and a low conducting solution in the pressure jump technique puts limitations on the type of studies that can be performed. For instance, reactions in the presence of very high 1 Present address: Chemistry Department, Laurentian University, Ontario, Canada. Reprint requests should be sent to this address. 273
Sudbury,
274
GOLDSACK,
HURST,
AND LOVE:
concentrations of organic solvents or even in pure organic solvents cannot be studied with the temperature jump technique, and the necessity for low salt concentrations in the pressure jump technique with conductivity detection precludes, for example, the study of the effects of high concentrations of such denaturants as guanidine chloride on protein systems. To overcome these limitations, a pressure jump apparatus with optical detection was developed and is described here.2 With this type of apparatus such studies as outlined above are possible. Preliminary kinetic results are also reported on the isomerization of bovine serum albumin at alkaline pH. Studies of this isomerization by the deuterium exchange (4) and optical rotation (5) methods have shown that this transition is dependent on pH as well as on type and concentration of salt present in solution. EXPERIMENTAL
A. Apparatus Design The design of the pressure bomb is given in Figure 1. It was found that the size of the sample cell was a critical parameter since an earlier version of the bomb with a cell height of 4 cm gave a large oscillatory effect with a decay time of about 250 psec. A Mylar disc of 0.005 in. thickness was used to separate the sample chamber from the oil pressure chamber. A very low viscosity oil (Imperial Oil Mentor 29) was found to be the best pressure transmitting medium because of ease of cleaning of the cell and a lower instrument time constant. Higher viscosity oil (White Paraffin Oil, Saybolt viscosity 125-135) was found to increase the instrument time constant slightly. Different bursting pressures were obtained with a variety of rupture discs, e.g., 0.003, 0.004, and 0.005 in. brass shim stock gave bursting pressures of 600, 950, and 1250 psi., respectively. A Power Packer double-action hand-operated hydraulic pump was used to slowly build up the pressure in the pressure chamber until the bursting disc broke spontaneously. The windows were made of Plexiglas and were found to become cloudy after about a thousand pressure drops. The pressure transducer was a model.LD80 obtained from Atlantic Research Corporation. It was used-only for .triggering slow phenomena since fast transients were more .easiiy -triggered on the internal trigger of the oscilloscope. ” B It has been kindly pointed out by a reviewer that a pressure jump apparatus with optical defection has been previously built (8). The apparatus described in the present paper, however, covers another time range than the apparatus of A. Jost.
OPTICAL
BRASS
275
APPAltATUS
DlSCp-m~m.
-?--
WASHER--
I-kXBROACHEO MYLAR
jtri\/p
WASHER ----7*,
RUPTURE TEFLON
PRESSURE
DISC
r
PLUGS-m-
-t
LENS ,--.--.-LENS
HOUSING BRASS
TO
FIG. 1. Cross-sectional
diagram
WASHER
SCALE
of pressure
bomb.
A Tetronix storage oscilloscope RM564 with type 3A7 and 2B67 plug-ins was used to store the transients temporarily ; a C-30 oscilloscope camera was employed for photographing the stored traces. A special high-intensity lamp (Astron Projection lamp 8V 50W) was found to be necessary because of the small size of the pressure bomb windows. The pressure bomb was fitted into a standard large cell holder of a Beckman DU monochromator. A circuit diagram of the detector unit is given in Figure 2. It was found that shorting out the last four stages of the photomultiplier gave a greatly increased signal-to-noise ratio. Typical results were 2 mV of noise with a 3 V signal output to the oscillo-
278
GOLDSACK, HURST, AND LOVE
FIG. 2. Circuit
diagram
of detector
unit of pressure
jump
apparatus.
scope. A RCA lP21 photomultiplier tube was used since most of the studies were conducted in the 520 to 560 mp region. The highvoltage power supply for the photomultiplier tube was a Fluke model 409 A. MATERIAL
AND
METHODS
Potassium nitrate and tris (hydroxymethylamino) methane (Tris) were reagent grade. Bovine serum albumin was purchased from Sigma Chemical Company and was used without further purification. Concentrations of the stock solution of the protein were estimated from absorbance readings at 280 rnp using a value of Et%.,, = 6.60 (4). The pH values of the solutions were measured with a Beckman zeromatic pH meter and were adjusted with stock KOH and HNOs solutions. All solutions were 0.2M in KNO, and O.OlM in Tris buffer. The phenol red and cresol red indicators were purchased from Fisher. The sample and pressure cells were thoroughly washed out with n-butunol followed by several flushings with distilled water before introduction of a new sample into the sample chamber. Great care
OPTICAL
PRESSURE
JUMP
APPARATUS
277
was taken to prevent oil from coming into contact with the solutions. At least three pictures were taken of every relaxation effect and the relaxation times evaluated from the Polaroid photographs in the usual manner from the slope of a plot of the logarithm of the amplitude decrement versus time. The time constant of the pressure jump apparatus was approximately 100 psec. It was measured by following the transient response of a solution of 2 X 1O-5 M cresol red indicator in 0.10 M Tris buffer at pH 8.2. Since the relaxation time for the indicatorhydrogen ion reaction is much less than 1 psec in such a highly buffered medium (1)) the observed transient decay must be due to mechanical causes. A typical picture of the response of the instrument in the 100 psec region is shown in Figure 3. RESULTS
Preliminary kinetic studies were carried out with this apparatus on the isomerization of bovine serum albumin which has been reported to occur in the region of pH 7 to 9 (4,5). Phenol red indicator was used as a means of coupling the isomerization to a color change so that the colorless isomerization could be detected (1). Two com-
FIG. 3. Relaxation effect due to mechanical relaxation of the pressure bomb. Sweep time was 100 psec/cm. Solution was 2X 10-S M in cresol red and 0.10 A4 in Tris buffer. pH was 8.2 A copper disc of 0.004 in thickness was used to obtain a pressure drop of 950 psi.
278
GOLDSACK,
HURST,
AND
LOVE
pletely separated relaxation effects were observed. The first relaxation time, called 71, was found to be independent of the concentration of protein and dye at any pH when the protein was varied from 1 X 10” to 1 X 1O-s M; it had low signal amplitudes, was pH dependent, and occurred in the 100 to 200 msec time region. The second relaxation time, called r2, was found to be dependent on the concentration of protein and dye, had large amplitudes, and occurred in the 2 to 10 msec time region. Typical photographs of these effects are given in Figure 4. To correlate the observed relaxation times 71 and sz with a kinetic mechanism, a model must be assumed. The simplest is the following, where P and P’ represents the folded and unfolded forms of the protein, respectively, D represents the indicator, and PD and P’D are complexes of the folded and unfolded forms of the protein with the indicator: 2 P
+
D
kl2
_
PD
k2l
(1) P’
+
D
k 34
_
P’D
k43 3
4
The small k’s represent individual rate constants for each step of the reaction. Mechanism 1 in general would have 3 relaxation times. However, not all 3 effects may be necessarily observed in any particular solution because of amplitude and time region limitations. The interpretation of the relaxation data for this type of system has been thoroughly discussed (6). Since r1 was found to be independent of concentration of both protein and indicator, it can be associated with the P to P’ isomerization reaction. Since 51 and Q are separated by at least a factor of 20, then 71 is related to the rate constants of mechanism 1 by (6) :
Equation 2 as expected is independent of concentration of protein and indicator, At pH 8.0, TV was found to be 250 & 25 msec and
OPTICAL
PRESSURE
JUMP
APPARATUS
2’49
FIG. 4. Relaxation effects observed in the bovine serum albumin-phenol red system: (A) Photograph of T?. Sweep time is 10 msec/cm and amplitude 60 mV/cm. Total concentration of protein was 2.9 X 10-G M, that of indicator 1.8 X 10-5 M, and the pH 7.0. (B) Photograph of T*. Sweep time is 100 msec/cm, amplitude 2 mV/cm, and the pH 8.5.
at pH 8.5, r1 was 180 =t 20 msec. These results imply that either oy both k,, and k,, are pH dependent. The ‘second relaxation time, TV, was dependent on the concentration of protein and dye. Preliminary data for 72 for several concentrations of protein and dye are given in Table 1. Since P is the
GOLDSACK, IIURST, Variation
AND LOVE
TABLE 1 of ze with Concentration
0.29 2.9 5.9
of Protein and Dye
1.8 1.8 1.6
7.5 6.5 5.7
zk 0.75 f 0.5 f 0.2
folded or native form of the protein at neutral pH and P’ the unfolded form at alkaline pH (4, 5)) then at pH 7.0 the path of the mechanism involving P’ with D can be neglected to a first approximation. In that case the mechanism becomes:
(3) P'
P'D
If 71 and TV are well separated
then the equation
for 72 is (6) : (4)
where P and fi are the equilibrium concentrations of the native protein and dye, respectively. Equilibrium dialysis experiments have been carried out with phenol red and bovine serum albumin (7)) and an apparent binding constant of 1.2 X 1O-5 M at pH 7.0 was obtained for binding of the dye to the protein. Defining KD as the binding constant determined from dialysis experiments, then: K
D
= F+ mm (PD + PD’) P-1
K D = (P)(D) (PD)
Utilizing viz. :
the conservation
(1 +F) l
(1 + P’D) PD
of mass relations
PO = i? + P, + PD + P’D
for protein
and dye, (7)
OPTICAL
PRESSURE
Do = n +i% and equation
5, a relation
281
APPARATUS
+P’D
for
(P + P’ + D) = +‘o
JUMP
(8)
(P + P + D) can be derived : + Do + Kr$
- 4PoDo
- KD
(9) If it is assumed that P > P’ at pH 7.0, then the data in Table 1 can be utilized to obtain a preliminary value of k,, and (k,, + k,, + k,,) from th e slope and intercept, respectively, of a plot of l/~ versus (P + D) . These values are found to be k,, = 1.4 X IO6 M-' sec.-l, and (k,, + k,, + kZ) = 1.1 X lo2 set-I, with an error of approximately 15 %. SUMMARY
A pressure jump apparatus with optical detection for studying fast transient chemical reactions has been described. The instrument can be used in the time region of 100 psec to 5 sec. Preliminary results on the kinetics of isomerization of bovine serum albumin at alkaline pH are reported. Two well-separated relaxation times were found. The slow relaxation time (100 to 200 msec) was concentration independent and was attributed, therefore, to the isomerization of the protein. The fast relaxation time (2 to 10 msec) was dependent on the concentration of protein and indicator (phenol red), and was associated with the binding of the dye to the protein which is coupled to the isomerization of the protein-dye complex. ACKNOWLEDGMENT The authors would like to thank the director of this station, Dr. H. L. A. Tarr, for his kind encouragement while we were carrying out this project. REFERENCES DE MAEYER, L., in “Techniques of Organic Chemistry” (A. ed.), Vol. 8, Part 2, p. 895. Interscience, New York, 1963. HAMMES, G. G., Advan. Enxymol. 25, 1 (1963). Ph.D. Thesis, University of Wisconsin, 1964. HALLAWAY, B. E., AND LUMRY, R. W., J. Bi01. C&m. 239,
1. EIGEN, M., AND Weissberger, 2. EIGEN, M., AND 3. TAKAHASHI, M., 4. BENSON, E. S., 122 (1964). 5. LEONARD, W. J., JR., VIJAI, K. K., AND FOSTER, J. F., J. Biol. Chem. 238, 1984 (1963). 6. ALBFXTY, R. A., YAGIL, G., DIVEN, W. F., AND TAKAHASHI, M., Acta Chem. Scand.17, S34 (1963). 7. RODKFX, F. L., Arch. Biochem. Biophys. 94, 38 (1961). 8. JOST, A., Ben. Bunsenges. Physikal. Chew. 70, 1057 (1966).
BIOCHEMISTBY
A Pressure
28,
Jump
Research
Board
(1969)
Apparatus
D. E. GOLDSACK’, Fisheties
273-281
of
with
R. E. HURST, Canada, Vancouver B. C., Canada
Received August
Optical AND
Detection J. LOVE
Laboratory,
Vancouver
8,
9, 1968
The recent introduction of perturbation techniques into chemical kinetic studies in solution has now made possible studies of chemical reactions whose reaction times fall in the second to microsecond region (1). These techniques have already been used to investigate fundamental biophysical problems such as the transient behavior of enzyme reactions and the isomerization of proteins (2,3). The essence of these perturbation techniques is to take a chemical system in a state of equilibrium and to quickly perturb the system to a new equilibrium state by a sudden change in an external variable such as temperature, pressure, or electric field strength (1). If the change in the external variable is fast enough (1 to 50 psec) , then chemical reactions which are slower than this change can be followed by appropriate monitoring techniques. For studies of protein systems the two methods applied so far have been the temperature jump technique (2) and the pressure jump method (3). The temperature jump technique involves creating a fast temperature rise in a highly conductive aqueous solution by joule heating, and the chemical change is monitored optically by following the small change in absorbance of the system. The pressure jump technique on the other hand involves application of a static pressure to an aqueous solution; a sharp drop in pressure is produced by a bursting membrane technique. In this case the system is monitored by following the small changes in conductivity of the solution. The requirement of a highly conductive solution in the temperature jump technique and a low conducting solution in the pressure jump technique puts limitations on the type of studies that can be performed. For instance, reactions in the presence of very high 1 Present address: Chemistry Department, Laurentian University, Ontario, Canada. Reprint requests should be sent to this address. 273
Sudbury,
274
GOLDSACK,
HURST,
AND LOVE:
concentrations of organic solvents or even in pure organic solvents cannot be studied with the temperature jump technique, and the necessity for low salt concentrations in the pressure jump technique with conductivity detection precludes, for example, the study of the effects of high concentrations of such denaturants as guanidine chloride on protein systems. To overcome these limitations, a pressure jump apparatus with optical detection was developed and is described here.2 With this type of apparatus such studies as outlined above are possible. Preliminary kinetic results are also reported on the isomerization of bovine serum albumin at alkaline pH. Studies of this isomerization by the deuterium exchange (4) and optical rotation (5) methods have shown that this transition is dependent on pH as well as on type and concentration of salt present in solution. EXPERIMENTAL
A. Apparatus Design The design of the pressure bomb is given in Figure 1. It was found that the size of the sample cell was a critical parameter since an earlier version of the bomb with a cell height of 4 cm gave a large oscillatory effect with a decay time of about 250 psec. A Mylar disc of 0.005 in. thickness was used to separate the sample chamber from the oil pressure chamber. A very low viscosity oil (Imperial Oil Mentor 29) was found to be the best pressure transmitting medium because of ease of cleaning of the cell and a lower instrument time constant. Higher viscosity oil (White Paraffin Oil, Saybolt viscosity 125-135) was found to increase the instrument time constant slightly. Different bursting pressures were obtained with a variety of rupture discs, e.g., 0.003, 0.004, and 0.005 in. brass shim stock gave bursting pressures of 600, 950, and 1250 psi., respectively. A Power Packer double-action hand-operated hydraulic pump was used to slowly build up the pressure in the pressure chamber until the bursting disc broke spontaneously. The windows were made of Plexiglas and were found to become cloudy after about a thousand pressure drops. The pressure transducer was a model.LD80 obtained from Atlantic Research Corporation. It was used-only for .triggering slow phenomena since fast transients were more .easiiy -triggered on the internal trigger of the oscilloscope. ” B It has been kindly pointed out by a reviewer that a pressure jump apparatus with optical defection has been previously built (8). The apparatus described in the present paper, however, covers another time range than the apparatus of A. Jost.
OPTICAL
BRASS
275
APPAltATUS
DlSCp-m~m.
-?--
WASHER--
I-kXBROACHEO MYLAR
jtri\/p
WASHER ----7*,
RUPTURE TEFLON
PRESSURE
DISC
r
PLUGS-m-
-t
LENS ,--.--.-LENS
HOUSING BRASS
TO
FIG. 1. Cross-sectional
diagram
WASHER
SCALE
of pressure
bomb.
A Tetronix storage oscilloscope RM564 with type 3A7 and 2B67 plug-ins was used to store the transients temporarily ; a C-30 oscilloscope camera was employed for photographing the stored traces. A special high-intensity lamp (Astron Projection lamp 8V 50W) was found to be necessary because of the small size of the pressure bomb windows. The pressure bomb was fitted into a standard large cell holder of a Beckman DU monochromator. A circuit diagram of the detector unit is given in Figure 2. It was found that shorting out the last four stages of the photomultiplier gave a greatly increased signal-to-noise ratio. Typical results were 2 mV of noise with a 3 V signal output to the oscillo-
278
GOLDSACK, HURST, AND LOVE
FIG. 2. Circuit
diagram
of detector
unit of pressure
jump
apparatus.
scope. A RCA lP21 photomultiplier tube was used since most of the studies were conducted in the 520 to 560 mp region. The highvoltage power supply for the photomultiplier tube was a Fluke model 409 A. MATERIAL
AND
METHODS
Potassium nitrate and tris (hydroxymethylamino) methane (Tris) were reagent grade. Bovine serum albumin was purchased from Sigma Chemical Company and was used without further purification. Concentrations of the stock solution of the protein were estimated from absorbance readings at 280 rnp using a value of Et%.,, = 6.60 (4). The pH values of the solutions were measured with a Beckman zeromatic pH meter and were adjusted with stock KOH and HNOs solutions. All solutions were 0.2M in KNO, and O.OlM in Tris buffer. The phenol red and cresol red indicators were purchased from Fisher. The sample and pressure cells were thoroughly washed out with n-butunol followed by several flushings with distilled water before introduction of a new sample into the sample chamber. Great care
OPTICAL
PRESSURE
JUMP
APPARATUS
277
was taken to prevent oil from coming into contact with the solutions. At least three pictures were taken of every relaxation effect and the relaxation times evaluated from the Polaroid photographs in the usual manner from the slope of a plot of the logarithm of the amplitude decrement versus time. The time constant of the pressure jump apparatus was approximately 100 psec. It was measured by following the transient response of a solution of 2 X 1O-5 M cresol red indicator in 0.10 M Tris buffer at pH 8.2. Since the relaxation time for the indicatorhydrogen ion reaction is much less than 1 psec in such a highly buffered medium (1)) the observed transient decay must be due to mechanical causes. A typical picture of the response of the instrument in the 100 psec region is shown in Figure 3. RESULTS
Preliminary kinetic studies were carried out with this apparatus on the isomerization of bovine serum albumin which has been reported to occur in the region of pH 7 to 9 (4,5). Phenol red indicator was used as a means of coupling the isomerization to a color change so that the colorless isomerization could be detected (1). Two com-
FIG. 3. Relaxation effect due to mechanical relaxation of the pressure bomb. Sweep time was 100 psec/cm. Solution was 2X 10-S M in cresol red and 0.10 A4 in Tris buffer. pH was 8.2 A copper disc of 0.004 in thickness was used to obtain a pressure drop of 950 psi.
278
GOLDSACK,
HURST,
AND
LOVE
pletely separated relaxation effects were observed. The first relaxation time, called 71, was found to be independent of the concentration of protein and dye at any pH when the protein was varied from 1 X 10” to 1 X 1O-s M; it had low signal amplitudes, was pH dependent, and occurred in the 100 to 200 msec time region. The second relaxation time, called r2, was found to be dependent on the concentration of protein and dye, had large amplitudes, and occurred in the 2 to 10 msec time region. Typical photographs of these effects are given in Figure 4. To correlate the observed relaxation times 71 and sz with a kinetic mechanism, a model must be assumed. The simplest is the following, where P and P’ represents the folded and unfolded forms of the protein, respectively, D represents the indicator, and PD and P’D are complexes of the folded and unfolded forms of the protein with the indicator: 2 P
+
D
kl2
_
PD
k2l
(1) P’
+
D
k 34
_
P’D
k43 3
4
The small k’s represent individual rate constants for each step of the reaction. Mechanism 1 in general would have 3 relaxation times. However, not all 3 effects may be necessarily observed in any particular solution because of amplitude and time region limitations. The interpretation of the relaxation data for this type of system has been thoroughly discussed (6). Since r1 was found to be independent of concentration of both protein and indicator, it can be associated with the P to P’ isomerization reaction. Since 51 and Q are separated by at least a factor of 20, then 71 is related to the rate constants of mechanism 1 by (6) :
Equation 2 as expected is independent of concentration of protein and indicator, At pH 8.0, TV was found to be 250 & 25 msec and
OPTICAL
PRESSURE
JUMP
APPARATUS
2’49
FIG. 4. Relaxation effects observed in the bovine serum albumin-phenol red system: (A) Photograph of T?. Sweep time is 10 msec/cm and amplitude 60 mV/cm. Total concentration of protein was 2.9 X 10-G M, that of indicator 1.8 X 10-5 M, and the pH 7.0. (B) Photograph of T*. Sweep time is 100 msec/cm, amplitude 2 mV/cm, and the pH 8.5.
at pH 8.5, r1 was 180 =t 20 msec. These results imply that either oy both k,, and k,, are pH dependent. The ‘second relaxation time, TV, was dependent on the concentration of protein and dye. Preliminary data for 72 for several concentrations of protein and dye are given in Table 1. Since P is the
GOLDSACK, IIURST, Variation
AND LOVE
TABLE 1 of ze with Concentration
0.29 2.9 5.9
of Protein and Dye
1.8 1.8 1.6
7.5 6.5 5.7
zk 0.75 f 0.5 f 0.2
folded or native form of the protein at neutral pH and P’ the unfolded form at alkaline pH (4, 5)) then at pH 7.0 the path of the mechanism involving P’ with D can be neglected to a first approximation. In that case the mechanism becomes:
(3) P'
P'D
If 71 and TV are well separated
then the equation
for 72 is (6) : (4)
where P and fi are the equilibrium concentrations of the native protein and dye, respectively. Equilibrium dialysis experiments have been carried out with phenol red and bovine serum albumin (7)) and an apparent binding constant of 1.2 X 1O-5 M at pH 7.0 was obtained for binding of the dye to the protein. Defining KD as the binding constant determined from dialysis experiments, then: K
D
= F+ mm (PD + PD’) P-1
K D = (P)(D) (PD)
Utilizing viz. :
the conservation
(1 +F) l
(1 + P’D) PD
of mass relations
PO = i? + P, + PD + P’D
for protein
and dye, (7)
OPTICAL
PRESSURE
Do = n +i% and equation
5, a relation
281
APPARATUS
+P’D
for
(P + P’ + D) = +‘o
JUMP
(8)
(P + P + D) can be derived : + Do + Kr$
- 4PoDo
- KD
(9) If it is assumed that P > P’ at pH 7.0, then the data in Table 1 can be utilized to obtain a preliminary value of k,, and (k,, + k,, + k,,) from th e slope and intercept, respectively, of a plot of l/~ versus (P + D) . These values are found to be k,, = 1.4 X IO6 M-' sec.-l, and (k,, + k,, + kZ) = 1.1 X lo2 set-I, with an error of approximately 15 %. SUMMARY
A pressure jump apparatus with optical detection for studying fast transient chemical reactions has been described. The instrument can be used in the time region of 100 psec to 5 sec. Preliminary results on the kinetics of isomerization of bovine serum albumin at alkaline pH are reported. Two well-separated relaxation times were found. The slow relaxation time (100 to 200 msec) was concentration independent and was attributed, therefore, to the isomerization of the protein. The fast relaxation time (2 to 10 msec) was dependent on the concentration of protein and indicator (phenol red), and was associated with the binding of the dye to the protein which is coupled to the isomerization of the protein-dye complex. ACKNOWLEDGMENT The authors would like to thank the director of this station, Dr. H. L. A. Tarr, for his kind encouragement while we were carrying out this project. REFERENCES DE MAEYER, L., in “Techniques of Organic Chemistry” (A. ed.), Vol. 8, Part 2, p. 895. Interscience, New York, 1963. HAMMES, G. G., Advan. Enxymol. 25, 1 (1963). Ph.D. Thesis, University of Wisconsin, 1964. HALLAWAY, B. E., AND LUMRY, R. W., J. Bi01. C&m. 239,
1. EIGEN, M., AND Weissberger, 2. EIGEN, M., AND 3. TAKAHASHI, M., 4. BENSON, E. S., 122 (1964). 5. LEONARD, W. J., JR., VIJAI, K. K., AND FOSTER, J. F., J. Biol. Chem. 238, 1984 (1963). 6. ALBFXTY, R. A., YAGIL, G., DIVEN, W. F., AND TAKAHASHI, M., Acta Chem. Scand.17, S34 (1963). 7. RODKFX, F. L., Arch. Biochem. Biophys. 94, 38 (1961). 8. JOST, A., Ben. Bunsenges. Physikal. Chew. 70, 1057 (1966).