ANALYTICAL
BIOCHEMISTRY
71, 186- 192 (1976)
A Microassay
for Lysyl Oxidase Activity’
RONALD L. MISIOROWSKI,JUDITH AND MILOS CHVAPIL Division
of Surgical Biology, Department of Surgery, Univeristy of Arizona, Tucson, Arizona
B. ULREICH College 85724
of Medicine,
Received May 1, 1975; accepted October 21, 1975 Pinnell and Martin’s [(1968) Proc. Nat. Acad. Sci. USA 61,708-7161 standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.
Lysyl oxidase oxidatively deaminates e-amino groups of peptide-bound hydroxylysine and lysine in collagen and lysine in elastin to the corresponding aldehydes. Aldol condensation or the reaction of aldehydes with e-amino groups of lysine or hydroxylysine to form Schiff bases then creates the nonenzymatically formed cross-links found in collagen and elastin. The assay developed by Pinnell and Martin (1) measures the enzymedependent release of tritium from the biosynthetically prepared 3Hlabeled substrate as lysine residues on the peptide are converted to aldehydes (Fig. 1). The released tritium equilibrates with water in the reaction vial and, after distillation, can be used as an indication of enzyme activity. In our attempts to purify lysyl oxidase we required an assay which was less cumbersome, time consuming, and expensive in the use of enzyme and substrate than that of Pinnell and Martin (1). Our modifications reduce consumption of materials by 8% and decrease the time spent in processing samples by 60-7%. PROCEDURES Preparation
of Labeled
Substrates
Insoluble elastin from 17-day chick embryo aortae and soluble collagen from chick embryo parietal bones were biosynthetically labeled with 4,5-3H lysine as described by Narayanan, Siegel and Martin (2). L-Lysine (4,5-3H), sp act 38.8 Ci/mmol, was obtained from New England Nuclear, Boston. 1 Supported in part by N.I.H.
grant Nos. AM 18706 and HL 16385.
186 Copyright D 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
LYSYL
OXIDASE
LO
1YSYl
(a) H-A-(CH,),--CH,-C3H,-NH3
oxidase
rlrH 3H+ + H,O
187
MiCROASSAY
_
.
I F=O H-C-(CH,)3-CH,-CC, rirH I
-
3HHO + H+
s
H-~-CH,-C3H3-C3H,-CC//o riiH
50
3H
+ 3Hf
L-o
Lo (b) H-A-CH,-C3H,-C3H,-CH,-NH, rlrH I
Lo
/OH +
eH-A-CH,--CPH,-y=C IkH I
3H
‘H
3H+ + H,O
-
‘H
3H+
3HHO + H+
FIG. 1. Release of tritium and production of tritiated water during aldehyde formation in (a) C-6 tritiated lysine and (b) C-4,5 tritiated lysine.
Preparation of Enzyme
Urea extractions of tissue from 17-day chick embryo calvaria, aortae, sterna, tibiae and femora in various stages of purification are used as enzyme sources (Method to be reported). Standard Assay
The method of Pinnell and Martin (1) was followed without modification except that no magnetic stirring bars were used in the distillation vessels. The Microdistillation
Apparatus
A solid aluminum block with 6 holes drilled to contain our reaction vials (10 x 30 mm, Arthur H. Thomas, Philadelphia) is located on a standard laboratory heating plate (Fig. 2). Each vial is half-covered by the block when inserted. A seventh, smaller hole in the block contains a thermometer, used as a gauge to maintain constant temperature. A platform holds the condensing apparatus, a stainless steel pan with a porcelain inset to hold scintillation vials in an upright position. The pan is filled with a dry ice-ethanol mixture. Serum stoppers into which reaction vials can be snapped have two openings - one a small needle (25 G %) to allow entering air to agitate the material to be distilled, and the other a large needle
188
MISIOROWSKI,
ULREICH
FIG. 2. Microdistillation
AND CHVAPIL
apparatus,
(1gG 1%) to direct the heated vapor toward the condenser (scintillation vial). Teflon tubing connects the larger needles in the serum stoppers to pieces of glass tubing which pass through No. 2 two-hole rubber stoppers into the lower portions of the collection vials in the dry ice bath. In each vial a second piece of tubing leads from just below the No. 2 rubber stopper to a vacuum pump. A 3-way glass valve is used to control the vacuum. Since some tritiated water may not condense in the cooled scintillation vials, a cold trap is placed in the line to prevent radioactive contamination of the vacuum pump. Functioning
of the Microdistillation
Apparatus
Vials in which the enzymatic reaction has reached completion are placed in the microdistillation apparatus and heated to 85°C under a partial vacuum. The stream of air entering through the needle serves to prevent bumping and increase the evaporation surface. The reaction mixture is distilled directly into scintillation vials which have been precooled in a dry ice-ethanol bath. Approximately 6 min is required for vials to dry. The vials are checked periodically and removed from their holes in the aluminum block as their contents become dry. Residual tritiated water is flushed from the lines by injecting 0.05 ml methanol through the inlet needle in the serum stopper. The methanol vaporizes immediately, and the vials are removed from the apparatus after the vacuum has been shut off at the 3-way valve. While one bank of six reaction vials is being distilled, a second bank is prepared by attaching six additional reaction vials and precooling the scintillation vials. When the distillation of the samples in one bank is complete, the vials, connecting tubing, and scintillation vials are lifted and swung aside. An identical set of vials, connecting tubing, and scintillation
LYSYL
OXIDASE
189
MICROASSAY
vials is swung into place, and distillation is started by the opening of the second 3-way valve. Before placing the next set of reactionand scintillation vials in the just-used apparatus the lines are flushed with methanol and aspirated to dryness. We have found no residual 3H counts in the lines following this treatment. Using alternate banks of vials, one technician can distill SO-60 vials/hr. Used reaction vials are generally discarded but can be washed for reuse. The Microassay
for Lysyl
Oxidase
All enzymes, substrates and other additions are prepared in phosphate buffered saline (PBS) (0.1 M KH2POI, 0.16 M NaCl, pH 7.7). The reaction takes place in small glass vials (10 x 30 mm) with Teflon lined screw caps. Usually 0.1 ml of substrate (500,000 dpm) is added to 0.2 ml enzyme preparation, and any other additions are limited to 0.1 ml. Toluene (0.01 ml) may be added to prevent bacterial growth. The air space above the reaction mixture is exchanged with 100% 0, before capping since O2 has been found to be a requirement of the reaction (2). The reaction mixture is incubated at 37°C for 3 hr in a water bath with constant agitation at 120 oscillations/min. The reaction is stopped by cooling the vials to 4°C. After distillation (as described) 10 ml of Aquasol (New England Nuclear, Boston) is added to the frozen distillate in the scintillation vials, which are then counted in a Beckman LS-250 scintillation counter. A computer program calculates dpm based on the observed cpm and a calibrated external standard. Activities are expressed as dpm/ml enzyme. Variations in substrate concentration in daily preparations are normalized by the computer program to yield dpm released per 500,000 dpm in the substrate. Comparison
of the Microassay
with the Standard
Assay
The data we obtain with the microassay are comparable to that found using the standard assay method (Fig. 3). In the example shown, the microassay yielded from 6-34% more dpm/ml of enzyme; the larger deviation between assay systems occurred in samples with greater activity. This may be accounted for by (a) reduced loss of materials from handling and pipetting, (b) reduced loss of materials, on glassware and tubing because of smaller size, (c) slightly different kinetics of reaction caused by different relative surface area of reaction vessels to volume of reactants during incubation, (d) slightly different reaction kinetics caused by different 0, concentrations in the reaction vials, or (e) some other factor in the reaction which differs slightly from that in the standard assay (under investigation). The microassay is suitable for kinetic studies. An example is the graph showing the effect of substrate concentration on reaction velocity (Fig. 4).
190
MISIOROWSKI,
ULREICH
AND CHVAPIL
12
microassay
-A-standard assay
F E6 EC 0a%6 --A P .z 2 b
4 2 10- lh+!f$T, 1
2
3 Tube
4 5 number
6
7
FIG. 3. A comparison of the microassay and the standard assay for lysyl oxidase during samples from a portion of a gel filtration column.
Both the microassay and the standard assay have coefficients of variation of less than 0.05. Advantages
of the Microassay
Table 1 compares the standard and microassay techniques for lysyl oxidase. The microassay saves time by reducing the total manipulation time per sample during distillation. Pipetting of 3HH0 from the condensing vessels into scintillation vials is eliminated, and washing of glassware is not necessary since the reaction vials are disposable. Considerable time and expense are spared in the preparation of enzymes and labeled substrate since only % as much material per assay is used in the microassay. If space is a consideration, 80 microassay reaction vials can be incubated in a small (8 x 23 cm) test tube rack, whereas 80 of the larger reaction vessels employed in the standard assay would fill several incubators and be unwieldy to handle.
6 0
1 SH-Lysine
2 SJbstrate
3 (dpm) ‘lo
-5
FIG. 4. Lineweaver-Burke plot of lysyl oxidase assayed with 3H-soluble collagen demonstrating the effect of substrate concentration on reaction velocity. Standard microassay conditions as described in Procedures were employed.
LYSYL
OXIDASE TABLE
COMPARWNS
191
MICROASSAY 1
OF ASSAY METHODS
FOR LYSYL
OXIDASE
Microassay Reaction vol
0.3-0.4 (a) 0. I (b) 0.2 (c) 0.1
Type of vacuum
Partial
Heat source for distillation
Aluminum
0, source
100% 0, for 2 secivial
Air in reaction vessel
Source of coofing for condensation
Ethanol-dry ice bath
Ethanol-dry ice bath
Distillation (mm)
10 x 30
28 x 200
vial size
Mixing device Condensing vial size
(mm)
ml ml substrate ml enzyme ml additions (optional)
Standard assay 1.5- 1.7 ml (a) 0.5 ml substrate (b) I .O ml enzyme (c) 0.2 ml additions (optional) Full
block on hot plate
Water bath
Air stream
Bar magnet
26 x 56 (standard scintillation vial)
28 x 200
Distillation (“a
temperature
85
SO-70
Distillation
time
6 mitt + time to swing duplicate apparatus into place (approx 5 set)
6 min + time to change reaction vessels and condensing vessels (approx 3-4 min)
% distillate counted
0.3-0.4 ml (100%)
0.5- I .O ml (29-67%)
Distillations run together (No.)
6
4
Samples distilled/hour (including changing of vessels, etc.) (No.)
50-60
24-28
Disadvantages of the Microassay
One additional step in the microassay is the necessity of exchanging the air in the reaction vial with O2 before incubation to assure the presence of enough 0, to allow the reaction to proceed normally. This step is especially important when 3H-insoluble elastin is used as substrate. It is assumed that the larger vessels used in the standard assay contain sufficient 0, because of their size. But since exchanging the air in 80 reaction vials requires less than 3 min (2 set/vial), the time involved is insignificant. The major drawback of the microassay is reduced recovery of 3HH0
192
MISIOROWSKI,
ULREICH
AND CHVAPIL
during distillation. When samples containing from 1 x IO3dpm to 2 x lo4 dpm are distilled in both apparati, the micromethod yields from 85 to 93% recovery of 3HH0 as compared to 93 to 100% recovery in the standard assay. Reasons for the decrease in efficiency using the microdistillation apparatus may be: (a) flushing residual tritiated water from the tubing with methanol may cause some carryover of 3HH0 by the vacuum pump into the cold trap (b) the use of a partial vacuum and the resulting stream of air over the distilling samples may cause some 3HH0 to pass through the condensing vial into the cold trap. SUMMARY
We find that in comparative studies of lysyl oxidase activity using the standard assay and the microassay described here, the final experimental results are similar despite differences in gross counts. Either system yields valid, useful results. For greater recovery of 3HH0, and thus, accuracy, the standard assay should be chosen, whereas the microassay is especially useful for screening large numbers of samples for activity and saves much expense and energy. ACKNOWLEDGMENTS The authors gratefully acknowledge and Bruce Mayall.
the skilled technical assistance of Linda Tillema
REFERENCES 1. Pinnell, S. R., and Martin, G. R. (1%8)Proc. 2. Narayanan, A. S., Siegel, R. C., and Martin, 162, 231-237.
Nat.
Acad.
Sci.
G. R. (1974) Arch.
USA 61,708-716. Biochem. Biophys.
BIOCHEMISTRY
71, 186- 192 (1976)
A Microassay
for Lysyl Oxidase Activity’
RONALD L. MISIOROWSKI,JUDITH AND MILOS CHVAPIL Division
of Surgical Biology, Department of Surgery, Univeristy of Arizona, Tucson, Arizona
B. ULREICH College 85724
of Medicine,
Received May 1, 1975; accepted October 21, 1975 Pinnell and Martin’s [(1968) Proc. Nat. Acad. Sci. USA 61,708-7161 standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.
Lysyl oxidase oxidatively deaminates e-amino groups of peptide-bound hydroxylysine and lysine in collagen and lysine in elastin to the corresponding aldehydes. Aldol condensation or the reaction of aldehydes with e-amino groups of lysine or hydroxylysine to form Schiff bases then creates the nonenzymatically formed cross-links found in collagen and elastin. The assay developed by Pinnell and Martin (1) measures the enzymedependent release of tritium from the biosynthetically prepared 3Hlabeled substrate as lysine residues on the peptide are converted to aldehydes (Fig. 1). The released tritium equilibrates with water in the reaction vial and, after distillation, can be used as an indication of enzyme activity. In our attempts to purify lysyl oxidase we required an assay which was less cumbersome, time consuming, and expensive in the use of enzyme and substrate than that of Pinnell and Martin (1). Our modifications reduce consumption of materials by 8% and decrease the time spent in processing samples by 60-7%. PROCEDURES Preparation
of Labeled
Substrates
Insoluble elastin from 17-day chick embryo aortae and soluble collagen from chick embryo parietal bones were biosynthetically labeled with 4,5-3H lysine as described by Narayanan, Siegel and Martin (2). L-Lysine (4,5-3H), sp act 38.8 Ci/mmol, was obtained from New England Nuclear, Boston. 1 Supported in part by N.I.H.
grant Nos. AM 18706 and HL 16385.
186 Copyright D 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
LYSYL
OXIDASE
LO
1YSYl
(a) H-A-(CH,),--CH,-C3H,-NH3
oxidase
rlrH 3H+ + H,O
187
MiCROASSAY
_
.
I F=O H-C-(CH,)3-CH,-CC, rirH I
-
3HHO + H+
s
H-~-CH,-C3H3-C3H,-CC//o riiH
50
3H
+ 3Hf
L-o
Lo (b) H-A-CH,-C3H,-C3H,-CH,-NH, rlrH I
Lo
/OH +
eH-A-CH,--CPH,-y=C IkH I
3H
‘H
3H+ + H,O
-
‘H
3H+
3HHO + H+
FIG. 1. Release of tritium and production of tritiated water during aldehyde formation in (a) C-6 tritiated lysine and (b) C-4,5 tritiated lysine.
Preparation of Enzyme
Urea extractions of tissue from 17-day chick embryo calvaria, aortae, sterna, tibiae and femora in various stages of purification are used as enzyme sources (Method to be reported). Standard Assay
The method of Pinnell and Martin (1) was followed without modification except that no magnetic stirring bars were used in the distillation vessels. The Microdistillation
Apparatus
A solid aluminum block with 6 holes drilled to contain our reaction vials (10 x 30 mm, Arthur H. Thomas, Philadelphia) is located on a standard laboratory heating plate (Fig. 2). Each vial is half-covered by the block when inserted. A seventh, smaller hole in the block contains a thermometer, used as a gauge to maintain constant temperature. A platform holds the condensing apparatus, a stainless steel pan with a porcelain inset to hold scintillation vials in an upright position. The pan is filled with a dry ice-ethanol mixture. Serum stoppers into which reaction vials can be snapped have two openings - one a small needle (25 G %) to allow entering air to agitate the material to be distilled, and the other a large needle
188
MISIOROWSKI,
ULREICH
FIG. 2. Microdistillation
AND CHVAPIL
apparatus,
(1gG 1%) to direct the heated vapor toward the condenser (scintillation vial). Teflon tubing connects the larger needles in the serum stoppers to pieces of glass tubing which pass through No. 2 two-hole rubber stoppers into the lower portions of the collection vials in the dry ice bath. In each vial a second piece of tubing leads from just below the No. 2 rubber stopper to a vacuum pump. A 3-way glass valve is used to control the vacuum. Since some tritiated water may not condense in the cooled scintillation vials, a cold trap is placed in the line to prevent radioactive contamination of the vacuum pump. Functioning
of the Microdistillation
Apparatus
Vials in which the enzymatic reaction has reached completion are placed in the microdistillation apparatus and heated to 85°C under a partial vacuum. The stream of air entering through the needle serves to prevent bumping and increase the evaporation surface. The reaction mixture is distilled directly into scintillation vials which have been precooled in a dry ice-ethanol bath. Approximately 6 min is required for vials to dry. The vials are checked periodically and removed from their holes in the aluminum block as their contents become dry. Residual tritiated water is flushed from the lines by injecting 0.05 ml methanol through the inlet needle in the serum stopper. The methanol vaporizes immediately, and the vials are removed from the apparatus after the vacuum has been shut off at the 3-way valve. While one bank of six reaction vials is being distilled, a second bank is prepared by attaching six additional reaction vials and precooling the scintillation vials. When the distillation of the samples in one bank is complete, the vials, connecting tubing, and scintillation vials are lifted and swung aside. An identical set of vials, connecting tubing, and scintillation
LYSYL
OXIDASE
189
MICROASSAY
vials is swung into place, and distillation is started by the opening of the second 3-way valve. Before placing the next set of reactionand scintillation vials in the just-used apparatus the lines are flushed with methanol and aspirated to dryness. We have found no residual 3H counts in the lines following this treatment. Using alternate banks of vials, one technician can distill SO-60 vials/hr. Used reaction vials are generally discarded but can be washed for reuse. The Microassay
for Lysyl
Oxidase
All enzymes, substrates and other additions are prepared in phosphate buffered saline (PBS) (0.1 M KH2POI, 0.16 M NaCl, pH 7.7). The reaction takes place in small glass vials (10 x 30 mm) with Teflon lined screw caps. Usually 0.1 ml of substrate (500,000 dpm) is added to 0.2 ml enzyme preparation, and any other additions are limited to 0.1 ml. Toluene (0.01 ml) may be added to prevent bacterial growth. The air space above the reaction mixture is exchanged with 100% 0, before capping since O2 has been found to be a requirement of the reaction (2). The reaction mixture is incubated at 37°C for 3 hr in a water bath with constant agitation at 120 oscillations/min. The reaction is stopped by cooling the vials to 4°C. After distillation (as described) 10 ml of Aquasol (New England Nuclear, Boston) is added to the frozen distillate in the scintillation vials, which are then counted in a Beckman LS-250 scintillation counter. A computer program calculates dpm based on the observed cpm and a calibrated external standard. Activities are expressed as dpm/ml enzyme. Variations in substrate concentration in daily preparations are normalized by the computer program to yield dpm released per 500,000 dpm in the substrate. Comparison
of the Microassay
with the Standard
Assay
The data we obtain with the microassay are comparable to that found using the standard assay method (Fig. 3). In the example shown, the microassay yielded from 6-34% more dpm/ml of enzyme; the larger deviation between assay systems occurred in samples with greater activity. This may be accounted for by (a) reduced loss of materials from handling and pipetting, (b) reduced loss of materials, on glassware and tubing because of smaller size, (c) slightly different kinetics of reaction caused by different relative surface area of reaction vessels to volume of reactants during incubation, (d) slightly different reaction kinetics caused by different 0, concentrations in the reaction vials, or (e) some other factor in the reaction which differs slightly from that in the standard assay (under investigation). The microassay is suitable for kinetic studies. An example is the graph showing the effect of substrate concentration on reaction velocity (Fig. 4).
190
MISIOROWSKI,
ULREICH
AND CHVAPIL
12
microassay
-A-standard assay
F E6 EC 0a%6 --A P .z 2 b
4 2 10- lh+!f$T, 1
2
3 Tube
4 5 number
6
7
FIG. 3. A comparison of the microassay and the standard assay for lysyl oxidase during samples from a portion of a gel filtration column.
Both the microassay and the standard assay have coefficients of variation of less than 0.05. Advantages
of the Microassay
Table 1 compares the standard and microassay techniques for lysyl oxidase. The microassay saves time by reducing the total manipulation time per sample during distillation. Pipetting of 3HH0 from the condensing vessels into scintillation vials is eliminated, and washing of glassware is not necessary since the reaction vials are disposable. Considerable time and expense are spared in the preparation of enzymes and labeled substrate since only % as much material per assay is used in the microassay. If space is a consideration, 80 microassay reaction vials can be incubated in a small (8 x 23 cm) test tube rack, whereas 80 of the larger reaction vessels employed in the standard assay would fill several incubators and be unwieldy to handle.
6 0
1 SH-Lysine
2 SJbstrate
3 (dpm) ‘lo
-5
FIG. 4. Lineweaver-Burke plot of lysyl oxidase assayed with 3H-soluble collagen demonstrating the effect of substrate concentration on reaction velocity. Standard microassay conditions as described in Procedures were employed.
LYSYL
OXIDASE TABLE
COMPARWNS
191
MICROASSAY 1
OF ASSAY METHODS
FOR LYSYL
OXIDASE
Microassay Reaction vol
0.3-0.4 (a) 0. I (b) 0.2 (c) 0.1
Type of vacuum
Partial
Heat source for distillation
Aluminum
0, source
100% 0, for 2 secivial
Air in reaction vessel
Source of coofing for condensation
Ethanol-dry ice bath
Ethanol-dry ice bath
Distillation (mm)
10 x 30
28 x 200
vial size
Mixing device Condensing vial size
(mm)
ml ml substrate ml enzyme ml additions (optional)
Standard assay 1.5- 1.7 ml (a) 0.5 ml substrate (b) I .O ml enzyme (c) 0.2 ml additions (optional) Full
block on hot plate
Water bath
Air stream
Bar magnet
26 x 56 (standard scintillation vial)
28 x 200
Distillation (“a
temperature
85
SO-70
Distillation
time
6 mitt + time to swing duplicate apparatus into place (approx 5 set)
6 min + time to change reaction vessels and condensing vessels (approx 3-4 min)
% distillate counted
0.3-0.4 ml (100%)
0.5- I .O ml (29-67%)
Distillations run together (No.)
6
4
Samples distilled/hour (including changing of vessels, etc.) (No.)
50-60
24-28
Disadvantages of the Microassay
One additional step in the microassay is the necessity of exchanging the air in the reaction vial with O2 before incubation to assure the presence of enough 0, to allow the reaction to proceed normally. This step is especially important when 3H-insoluble elastin is used as substrate. It is assumed that the larger vessels used in the standard assay contain sufficient 0, because of their size. But since exchanging the air in 80 reaction vials requires less than 3 min (2 set/vial), the time involved is insignificant. The major drawback of the microassay is reduced recovery of 3HH0
192
MISIOROWSKI,
ULREICH
AND CHVAPIL
during distillation. When samples containing from 1 x IO3dpm to 2 x lo4 dpm are distilled in both apparati, the micromethod yields from 85 to 93% recovery of 3HH0 as compared to 93 to 100% recovery in the standard assay. Reasons for the decrease in efficiency using the microdistillation apparatus may be: (a) flushing residual tritiated water from the tubing with methanol may cause some carryover of 3HH0 by the vacuum pump into the cold trap (b) the use of a partial vacuum and the resulting stream of air over the distilling samples may cause some 3HH0 to pass through the condensing vial into the cold trap. SUMMARY
We find that in comparative studies of lysyl oxidase activity using the standard assay and the microassay described here, the final experimental results are similar despite differences in gross counts. Either system yields valid, useful results. For greater recovery of 3HH0, and thus, accuracy, the standard assay should be chosen, whereas the microassay is especially useful for screening large numbers of samples for activity and saves much expense and energy. ACKNOWLEDGMENTS The authors gratefully acknowledge and Bruce Mayall.
the skilled technical assistance of Linda Tillema
REFERENCES 1. Pinnell, S. R., and Martin, G. R. (1%8)Proc. 2. Narayanan, A. S., Siegel, R. C., and Martin, 162, 231-237.
Nat.
Acad.
Sci.
G. R. (1974) Arch.
USA 61,708-716. Biochem. Biophys.
