NOTES
351
& TIPS
The Construction and Use of an Inexpensive Data Collection System for High-Resolution Chromatography’ Jerry M. Merz2 and Andrew J. Mort Department of Biochemistry and Molecular Agricultural Experiment Station, Oklahoma Stillwater, Oklahoma 74078-0454
FIG. 2.
Autoradiograph of a sequencing gel in which the performances of single-stranded DNA binding proteins added at different steps of the sequencing reaction were compared. All the reactions were stopped using modified stop mix. Track 1, SSBP added at the template denaturation step; Track 2, SSBP added at the labeling step; Track 3, T4 g32 added at the template denaturation step; Track 4, T4 g32 added at the labeling step. The arrowheads indicate premature enzyme pausing. The asterisk indicates resolution of this effect. All tracks were loaded in the order GATC.
SSBP at the template denaturation step resulted in enzyme “read-through.” Addition of g32 at this step had no effect, the most likely reason for this being protein denaturation. Removal of SSBP and g32 from sequencing reactions has previously been carried out by proteinase K digestion (1). Here we have described a simple modification of the sequencing stop solution using guanidinium isothiocyanate which effectively destroys the protein prior to electrophoresis (7). However, for this to be effective we used bovine serum albumin-free enzyme dilution buffer. The modifications described here complement other methods used to solve sequencing artifacts and problems. Acknowledgments. script. search
We thank Kathy Rogers This work was supported by a grant Council AIDS Directed Programme.
for typing the manufrom the Medical Re-
REFERENCES 1. Kaspar, P., Zadril, S., and Febry, M. (1989) Nucleic Acids Res. 17, 3616. 2. Winship, P. R. (1989) Nucleic Acids Res. 17, 1266. 3. United Bioscience (1991) Molecular Reagents Catalogue, p. 56. 4. Simmonds, P., Balfe, P., Peutherer, J. F., Ludlam, C. A., Bishop, J. O., and Leigh-Brown, A. J. (1991) J. Viral. 64, 5840-5850. 5. Ball, J. K., Whitwell, H., and Desselberger, U. (1991) Lancet 338(n), 63. 6. Tsang, T. C., and Bentley, D. R. (1988) Nucleic Acids Res. 13, 6238. 7. Sambrook, J., Fritsch, Cloning, 2nd ed. Cold Spring Harbor, NY.
E., and Maniatis, T. (1989) Molecular Spring Harbor Laboratory Press, Cold
Biology, Oklahoma State University,
Integrators such as those supplied by Spectra Physics offer real-time data analysis but, until recently, would not store the chromatograms for reprocessing with altered integrator parameters and would not export data for further analysis or processing. We realized that a more flexible system would be advantageous and ultimately cheaper if it were able to process data from several different instruments. Within the past few years the electronic industry has developed off-the-shelf components that are aimed primarily at the industrial control and smart appliances markets and that seem suitable for data loggers. A review of the literature has revealed no publications on the application of these components as networked data loggers for chromatographic systems-at least in a configuration that is easily assembled. We, therefore, built a system that incorporates some of these components and has the following features: simple construction, high dynamic range, low cost, programmability, expandability, networking capability, portability, fast processing, data archiving, and flexible data export. Materials and methods: Data logger. The data logger consists of three major hardware components (Alpha Products, Darien, CT): the microcomputer (SP-127), the analog to digital (A/D) converter (AN146), and the digital input/output (I/O) interface (DG-148). These three components communicate over a common bus. The microcomputer program running on the SP-127 controls the activity of the other components. The A/D converter receives the analog signal from the chromatograph through a programmable-gain-instrumentation amplifier (PGA201, Burr-Brown Corp., Tucson, AZ). The gain of this amplifier is controlled by the microcomputer through the I/O interface. A trigger signal from the chromatograph sensed through the I/O interface initiates data collection by the microcomputer. Alternatively, the user can control the function of the data logger through the buttons on its control panel. The microcomputer reports its current activity on a liquid i Supported by U.S. Department of Energy Grant DE-FG0586ER.13496 and the Oklahoma Agricultural Experiment Station, Oklahoma State University. This is Paper 6252 of the Oklahoma Agricultural Experiment Station. 2 To whom correspondence should be addressed. Parts list and program listings are available upon request.
207,351-353
(1992) 0003-2697/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
ANALYTICAL
BIOCHEMISTRY
352
NOTES
crystal display (LCD Model LTNlllR-10, Amperex Electronic CO., Smithfield, RI) driven by the I/O interface. The microcomputer’s memory stores newly collected data until they are automatically transferred to another computer for permanent storage and processing. The data transfer between the main computer (Apple Macintosh Plus) and the data logger is through a serial port set to operate in a local area network (LAN). This allows 10 or more data loggers to communicate with the main computer using the RS232 communication standard at 19,200-baud rate. Since the data logger has battery-supported memory (Smart Socket IC battery, Dallas Semiconductor, Dallas, TX), it can be disconnected from the LAN and transported to even greater distances to and from a signal source without data loss. Local area network. A standard round or flat telephone cable made of solid copper (24-guage four-conductor cable) is used for the LAN. The LAN can extend up to 300 m (- 1000 feet) depending on the number of data loggers connected to the cable. In addition to the wire, the LAN consists of readily available telephone components such as I&J11 4 conductors plugs, R.Jll duplex jacks with inline couplers, four-wire TEE adaptors, and an eight-pin mini-DIN plug connector for the Macintosh. Our LAN requires the use of a termination resistor at its extreme end and diodes at each node. The data logger’s Transmit Data line (TxD) is connected to the Macintosh Receive Data (-) line (RxD-) through a diode (general purpose, 50 V, 1 amp). This is to prevent the data logger’s TxD line from driving the main computer’s RxD line to a low state and preventing signals from other data loggers from being detected. The diode’s cathode should be directed away from the remote unit and toward the Macintosh. A single termination carbon resistor (1.5 K, 10%) is used to maintain a low state across the ground line and the transmit data line at the end of the network. Data logger program. The remote’s program was created in Basic-52 (1). The program’s design is based on two event loops: a wait loop and a command loop. The wait loop processes data acquisition and user buttons &vents and responds to the remote’s unique identification code sent to it by the central computer. The remote ignores all other signals on the LAN while in this loop. Once it receives its ID code, the program drops into the command loop. Here, it continues to process data acquisition events, user events, and also commands from the central computer. After receiving an exit command, the program flow returns to the wait loop. The data read from the A/D board are stored in memory as 2 bytes. The low byte contains the first 8 bits of data; the high byte contains the upper 4 bits (total of 12 bits for the voltage-an integer value of O-4096), the polarity (1 bit), and the gain setting (2 bits). This 2-byte
& TIPS
number is stored in the external memory of the CPU board in the area between the program variables’ space and the designated pseudo-ROM space where the program resides. These 2-byte numbers are subsequently transferred to the central computer (i.e., Macintosh) and deciphered into a floating point number representing the actual voltage. The maximum voltage range is k4.096 V, and the lowest range at the highest gain of 512 is f8.0 mV with a resolution of 1.953 pV. This represents a full-scale resolution of 0.00005%, or about the same dynamic range that would be given by a 21-bit A/D converter. The program controls the gain setting of the instrumentation amplifier (PGA 201) via one of the ports of the I/O board (DG-148). This is accomplishedby testing the first 4 bits of the high byte of each new data point as it is collected. When this value is greater than about 80% of full scale, the gain is reduced. If this value is less than about 8% of full scale, the gain is increased. The current data point is stored in RAM, and the next one is collected at the new gain setting. The binary gain of the amplifier (i.e., 1,8,64,512) supplies an effective hysteresis that prevents oscillations across any two gain settings. The display shows the voltage and polarity during data collection. In addition, the program uses the display as a one-line editor so that the remote’s name and parameters can be changed. The user can set the trigger state (on/off), the delay period (0 to 400 s), the rate of data collection (one, two, and four samples per second), and the number of data points to be collected (1000 to 15,000) by using the panel buttons. Central computer program. The main program allows the user to control the remote from a list of menu items that are logically dependent on the status of the remote. These functions are start data collection, start collecting now (override trigger and delay settings), store data, and stop data collection. The user can request the status of a remote, send a command to a remote, or look at the data being collected in real time either numerically or graphically. The program was created in Basic (2) and operates under the Apple Macintosh MultiFinder (3) disk operating system (version 6.0.2 or greater) and Macintosh Plus or later computer. It is compatible with most other programs and requires a 200K memory partition. The user can select the interval at which the program will poll the remotes. Initially this is set to 2 min. If the program finds that a remote is ready to transfer its data, it informs the user by its speech synthesizer that it has taken control of the main computer and then transfers the data from the remote. The 2 bytes per data point that the remote sends are translated into a signed floating point number and saved in a text file in the appropriate format for subse-
NOTES
quent processing by the integration program described below. The program uses the root directory to find the data folder that has the remote’s name. If it does not find the folder, it creates a new folder and places the data file within it. The files from the same remotes are distinguished from one another by a serial suffix number. The integration program is Analog Connection Chrom written by Drew Scientific and distributed by Strawberry Tree Computers, Inc. (Sunnyvale, CA). It can handle numeric ASCII files with up to 18,000 data points and accommodate up to 400 chromatographic peaks. In addition to peak recognition, the program allows for detector noise rejection, compensation for partially resolved and trailing edge peaks, tangent skimming, and calibration based on internal or external standards. The integration and other data analysis are done on another computer (Apple Macintosh 11x) so that the control program can operate without interrupting users. Data stored by the control program on its computer (Apple Macintosh Plus) are transferred to the other computer for processing via the AppleTalk network using MacTops (Sitka Corp., Alameda, CA) peerto-peer file server. Figure 1 illustrates a typical high-resolution chromatogram containing both large and small peaks of interest captured by the data logger. Summary. In recent years a cottage industry of suppliers of microcontrollers and auxiliary circuitry has developed which makes it practical to build data loggers much in the same manner as researchers develop a new chromatographic instrument from an assemblage of parts from various sources. We have built a system of
3.00 2.50 2
2.00 -
I = 1.50 8. d
1.00 0.50 0.00 -I
J I
0
IO
7
I
20
I
I
I
30
40
50
353
& TIPS
high-resolution data loggers that automatically collect and store data from a variety of chromatographic instruments and transmit the data to a microcomputer for integration and further analysis. This system allows more than 10 data loggers on a three-wire network to collect up to 15,000 data points at the equivalent resolution of a 21-bit A/D converter. The data loggers are easily transported, have long-term data storage, and have functioned reliably in our laboratory for the last 2 years. REFERENCES 1. Anonymous Santa Clara,
(1989) MCS CA 95051.
BASIC-52
User’s
Manual,
Intel
Corp.,
2. Gariepy, A. R. (1988) Zbasic for the Macintosh, ZEDCOR, son, AZ. 3. Anonymous (1989) Apple Computer, Inc., Inside Macintosh, 1, Addison-Wesley, New York. 4. Komalavilas, P., and Mort, A. J. (1989) Carbohydr. Res. 261-272.
TucVol. 189,
Preparation of Fully Oxidized Active and Reduced Inactive Forms of Galactose Oxidase from Dactylium dendroides Using Ferricyanide-Containing Oxidizing and Ferrocyanide-Containing Reducing Forms of Ion Exchange Resins’ Michael P. Montague-Smith, and Bruce P. Branchaud*
Rebekka
M. Wachter,
*Department of Chemistry, and Institute of Molecular University of Oregon, Eugene, Oregon 97403
Biology,
Galactose oxidase (EC 1.1.3.9) is a type II mononuclear copper protein secreted by the fungus Dactylium dendrokb2 The enzyme catalyzes the oxidation of primary alcohols with O,, producing aldehydes and H,O, (1). The details of the catalytic mechanism have not been fully elucidated. A chronic problem in kinetic assays of galactose oxidase is the tendency of the enzyme to exist as a mixture of oxidized active and one-electron reduced inactive forms (2-4). The two forms cannot be physically separated by standard purification techniques.
Time (min)
FIG. 1. A chromatogram collected by the data logger without any preprocessing (i.e., smoothing or filtering). The inset figure magnifies the peaks of most interest within the 14- to 20-min elution period. Even these and smaller peaks can be routinely and accurately integrated. The data represent a sample prepared from the methanolysis and derivation of cell-wall samples from cotton suspension cultures that was injected onto a capillary column installed in a Tracer 560 gas-liquid chromatograph that used a flame ionization detector. The derivatives were prepared as described by Komalavilas and Mort (4).
i This work was funded in part by Grant NSF MCB 91-18911 from the National Science Foundation, by Fellowship P20010238 from the U.S. Department of Education Graduate Assistance in Areas of National Need Program (M.M.-S.), and by Predoctoral Training Grant NIH GM07759 from the National Institutes of Health (R.M.W.). ’ Dactylium dendroides (Northern Regional Research Laboratories No. 2993, Peoria, IL.) was a kind gift from Dr. Daniel Kosman, Department of Biochemistry, State University of New York, Buffalo, NY. ANALYTICAL
BIOCHEMISTRY
207,353-355 0003-2697192
(1%X2) $5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reoroduction in anv form reserved.
351
& TIPS
The Construction and Use of an Inexpensive Data Collection System for High-Resolution Chromatography’ Jerry M. Merz2 and Andrew J. Mort Department of Biochemistry and Molecular Agricultural Experiment Station, Oklahoma Stillwater, Oklahoma 74078-0454
FIG. 2.
Autoradiograph of a sequencing gel in which the performances of single-stranded DNA binding proteins added at different steps of the sequencing reaction were compared. All the reactions were stopped using modified stop mix. Track 1, SSBP added at the template denaturation step; Track 2, SSBP added at the labeling step; Track 3, T4 g32 added at the template denaturation step; Track 4, T4 g32 added at the labeling step. The arrowheads indicate premature enzyme pausing. The asterisk indicates resolution of this effect. All tracks were loaded in the order GATC.
SSBP at the template denaturation step resulted in enzyme “read-through.” Addition of g32 at this step had no effect, the most likely reason for this being protein denaturation. Removal of SSBP and g32 from sequencing reactions has previously been carried out by proteinase K digestion (1). Here we have described a simple modification of the sequencing stop solution using guanidinium isothiocyanate which effectively destroys the protein prior to electrophoresis (7). However, for this to be effective we used bovine serum albumin-free enzyme dilution buffer. The modifications described here complement other methods used to solve sequencing artifacts and problems. Acknowledgments. script. search
We thank Kathy Rogers This work was supported by a grant Council AIDS Directed Programme.
for typing the manufrom the Medical Re-
REFERENCES 1. Kaspar, P., Zadril, S., and Febry, M. (1989) Nucleic Acids Res. 17, 3616. 2. Winship, P. R. (1989) Nucleic Acids Res. 17, 1266. 3. United Bioscience (1991) Molecular Reagents Catalogue, p. 56. 4. Simmonds, P., Balfe, P., Peutherer, J. F., Ludlam, C. A., Bishop, J. O., and Leigh-Brown, A. J. (1991) J. Viral. 64, 5840-5850. 5. Ball, J. K., Whitwell, H., and Desselberger, U. (1991) Lancet 338(n), 63. 6. Tsang, T. C., and Bentley, D. R. (1988) Nucleic Acids Res. 13, 6238. 7. Sambrook, J., Fritsch, Cloning, 2nd ed. Cold Spring Harbor, NY.
E., and Maniatis, T. (1989) Molecular Spring Harbor Laboratory Press, Cold
Biology, Oklahoma State University,
Integrators such as those supplied by Spectra Physics offer real-time data analysis but, until recently, would not store the chromatograms for reprocessing with altered integrator parameters and would not export data for further analysis or processing. We realized that a more flexible system would be advantageous and ultimately cheaper if it were able to process data from several different instruments. Within the past few years the electronic industry has developed off-the-shelf components that are aimed primarily at the industrial control and smart appliances markets and that seem suitable for data loggers. A review of the literature has revealed no publications on the application of these components as networked data loggers for chromatographic systems-at least in a configuration that is easily assembled. We, therefore, built a system that incorporates some of these components and has the following features: simple construction, high dynamic range, low cost, programmability, expandability, networking capability, portability, fast processing, data archiving, and flexible data export. Materials and methods: Data logger. The data logger consists of three major hardware components (Alpha Products, Darien, CT): the microcomputer (SP-127), the analog to digital (A/D) converter (AN146), and the digital input/output (I/O) interface (DG-148). These three components communicate over a common bus. The microcomputer program running on the SP-127 controls the activity of the other components. The A/D converter receives the analog signal from the chromatograph through a programmable-gain-instrumentation amplifier (PGA201, Burr-Brown Corp., Tucson, AZ). The gain of this amplifier is controlled by the microcomputer through the I/O interface. A trigger signal from the chromatograph sensed through the I/O interface initiates data collection by the microcomputer. Alternatively, the user can control the function of the data logger through the buttons on its control panel. The microcomputer reports its current activity on a liquid i Supported by U.S. Department of Energy Grant DE-FG0586ER.13496 and the Oklahoma Agricultural Experiment Station, Oklahoma State University. This is Paper 6252 of the Oklahoma Agricultural Experiment Station. 2 To whom correspondence should be addressed. Parts list and program listings are available upon request.
207,351-353
(1992) 0003-2697/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
ANALYTICAL
BIOCHEMISTRY
352
NOTES
crystal display (LCD Model LTNlllR-10, Amperex Electronic CO., Smithfield, RI) driven by the I/O interface. The microcomputer’s memory stores newly collected data until they are automatically transferred to another computer for permanent storage and processing. The data transfer between the main computer (Apple Macintosh Plus) and the data logger is through a serial port set to operate in a local area network (LAN). This allows 10 or more data loggers to communicate with the main computer using the RS232 communication standard at 19,200-baud rate. Since the data logger has battery-supported memory (Smart Socket IC battery, Dallas Semiconductor, Dallas, TX), it can be disconnected from the LAN and transported to even greater distances to and from a signal source without data loss. Local area network. A standard round or flat telephone cable made of solid copper (24-guage four-conductor cable) is used for the LAN. The LAN can extend up to 300 m (- 1000 feet) depending on the number of data loggers connected to the cable. In addition to the wire, the LAN consists of readily available telephone components such as I&J11 4 conductors plugs, R.Jll duplex jacks with inline couplers, four-wire TEE adaptors, and an eight-pin mini-DIN plug connector for the Macintosh. Our LAN requires the use of a termination resistor at its extreme end and diodes at each node. The data logger’s Transmit Data line (TxD) is connected to the Macintosh Receive Data (-) line (RxD-) through a diode (general purpose, 50 V, 1 amp). This is to prevent the data logger’s TxD line from driving the main computer’s RxD line to a low state and preventing signals from other data loggers from being detected. The diode’s cathode should be directed away from the remote unit and toward the Macintosh. A single termination carbon resistor (1.5 K, 10%) is used to maintain a low state across the ground line and the transmit data line at the end of the network. Data logger program. The remote’s program was created in Basic-52 (1). The program’s design is based on two event loops: a wait loop and a command loop. The wait loop processes data acquisition and user buttons &vents and responds to the remote’s unique identification code sent to it by the central computer. The remote ignores all other signals on the LAN while in this loop. Once it receives its ID code, the program drops into the command loop. Here, it continues to process data acquisition events, user events, and also commands from the central computer. After receiving an exit command, the program flow returns to the wait loop. The data read from the A/D board are stored in memory as 2 bytes. The low byte contains the first 8 bits of data; the high byte contains the upper 4 bits (total of 12 bits for the voltage-an integer value of O-4096), the polarity (1 bit), and the gain setting (2 bits). This 2-byte
& TIPS
number is stored in the external memory of the CPU board in the area between the program variables’ space and the designated pseudo-ROM space where the program resides. These 2-byte numbers are subsequently transferred to the central computer (i.e., Macintosh) and deciphered into a floating point number representing the actual voltage. The maximum voltage range is k4.096 V, and the lowest range at the highest gain of 512 is f8.0 mV with a resolution of 1.953 pV. This represents a full-scale resolution of 0.00005%, or about the same dynamic range that would be given by a 21-bit A/D converter. The program controls the gain setting of the instrumentation amplifier (PGA 201) via one of the ports of the I/O board (DG-148). This is accomplishedby testing the first 4 bits of the high byte of each new data point as it is collected. When this value is greater than about 80% of full scale, the gain is reduced. If this value is less than about 8% of full scale, the gain is increased. The current data point is stored in RAM, and the next one is collected at the new gain setting. The binary gain of the amplifier (i.e., 1,8,64,512) supplies an effective hysteresis that prevents oscillations across any two gain settings. The display shows the voltage and polarity during data collection. In addition, the program uses the display as a one-line editor so that the remote’s name and parameters can be changed. The user can set the trigger state (on/off), the delay period (0 to 400 s), the rate of data collection (one, two, and four samples per second), and the number of data points to be collected (1000 to 15,000) by using the panel buttons. Central computer program. The main program allows the user to control the remote from a list of menu items that are logically dependent on the status of the remote. These functions are start data collection, start collecting now (override trigger and delay settings), store data, and stop data collection. The user can request the status of a remote, send a command to a remote, or look at the data being collected in real time either numerically or graphically. The program was created in Basic (2) and operates under the Apple Macintosh MultiFinder (3) disk operating system (version 6.0.2 or greater) and Macintosh Plus or later computer. It is compatible with most other programs and requires a 200K memory partition. The user can select the interval at which the program will poll the remotes. Initially this is set to 2 min. If the program finds that a remote is ready to transfer its data, it informs the user by its speech synthesizer that it has taken control of the main computer and then transfers the data from the remote. The 2 bytes per data point that the remote sends are translated into a signed floating point number and saved in a text file in the appropriate format for subse-
NOTES
quent processing by the integration program described below. The program uses the root directory to find the data folder that has the remote’s name. If it does not find the folder, it creates a new folder and places the data file within it. The files from the same remotes are distinguished from one another by a serial suffix number. The integration program is Analog Connection Chrom written by Drew Scientific and distributed by Strawberry Tree Computers, Inc. (Sunnyvale, CA). It can handle numeric ASCII files with up to 18,000 data points and accommodate up to 400 chromatographic peaks. In addition to peak recognition, the program allows for detector noise rejection, compensation for partially resolved and trailing edge peaks, tangent skimming, and calibration based on internal or external standards. The integration and other data analysis are done on another computer (Apple Macintosh 11x) so that the control program can operate without interrupting users. Data stored by the control program on its computer (Apple Macintosh Plus) are transferred to the other computer for processing via the AppleTalk network using MacTops (Sitka Corp., Alameda, CA) peerto-peer file server. Figure 1 illustrates a typical high-resolution chromatogram containing both large and small peaks of interest captured by the data logger. Summary. In recent years a cottage industry of suppliers of microcontrollers and auxiliary circuitry has developed which makes it practical to build data loggers much in the same manner as researchers develop a new chromatographic instrument from an assemblage of parts from various sources. We have built a system of
3.00 2.50 2
2.00 -
I = 1.50 8. d
1.00 0.50 0.00 -I
J I
0
IO
7
I
20
I
I
I
30
40
50
353
& TIPS
high-resolution data loggers that automatically collect and store data from a variety of chromatographic instruments and transmit the data to a microcomputer for integration and further analysis. This system allows more than 10 data loggers on a three-wire network to collect up to 15,000 data points at the equivalent resolution of a 21-bit A/D converter. The data loggers are easily transported, have long-term data storage, and have functioned reliably in our laboratory for the last 2 years. REFERENCES 1. Anonymous Santa Clara,
(1989) MCS CA 95051.
BASIC-52
User’s
Manual,
Intel
Corp.,
2. Gariepy, A. R. (1988) Zbasic for the Macintosh, ZEDCOR, son, AZ. 3. Anonymous (1989) Apple Computer, Inc., Inside Macintosh, 1, Addison-Wesley, New York. 4. Komalavilas, P., and Mort, A. J. (1989) Carbohydr. Res. 261-272.
TucVol. 189,
Preparation of Fully Oxidized Active and Reduced Inactive Forms of Galactose Oxidase from Dactylium dendroides Using Ferricyanide-Containing Oxidizing and Ferrocyanide-Containing Reducing Forms of Ion Exchange Resins’ Michael P. Montague-Smith, and Bruce P. Branchaud*
Rebekka
M. Wachter,
*Department of Chemistry, and Institute of Molecular University of Oregon, Eugene, Oregon 97403
Biology,
Galactose oxidase (EC 1.1.3.9) is a type II mononuclear copper protein secreted by the fungus Dactylium dendrokb2 The enzyme catalyzes the oxidation of primary alcohols with O,, producing aldehydes and H,O, (1). The details of the catalytic mechanism have not been fully elucidated. A chronic problem in kinetic assays of galactose oxidase is the tendency of the enzyme to exist as a mixture of oxidized active and one-electron reduced inactive forms (2-4). The two forms cannot be physically separated by standard purification techniques.
Time (min)
FIG. 1. A chromatogram collected by the data logger without any preprocessing (i.e., smoothing or filtering). The inset figure magnifies the peaks of most interest within the 14- to 20-min elution period. Even these and smaller peaks can be routinely and accurately integrated. The data represent a sample prepared from the methanolysis and derivation of cell-wall samples from cotton suspension cultures that was injected onto a capillary column installed in a Tracer 560 gas-liquid chromatograph that used a flame ionization detector. The derivatives were prepared as described by Komalavilas and Mort (4).
i This work was funded in part by Grant NSF MCB 91-18911 from the National Science Foundation, by Fellowship P20010238 from the U.S. Department of Education Graduate Assistance in Areas of National Need Program (M.M.-S.), and by Predoctoral Training Grant NIH GM07759 from the National Institutes of Health (R.M.W.). ’ Dactylium dendroides (Northern Regional Research Laboratories No. 2993, Peoria, IL.) was a kind gift from Dr. Daniel Kosman, Department of Biochemistry, State University of New York, Buffalo, NY. ANALYTICAL
BIOCHEMISTRY
207,353-355 0003-2697192
(1%X2) $5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reoroduction in anv form reserved.
