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Lysozyme oligomers as a molecular mass standard for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
ANALYTICAL
171,419-422
BIOCHEMISTRY
Lysozyme
(1988)
Oligomers as a Molecular Sulfate-Polyacrylamide
Mass Standard for Sodium Dodecyl Gel Electrophoresis
RYSZARD DRO~D~ AND JERZY W. NASKALSKI Department
of Biochemical
Diagnostics,
Medical
Academy,
Krakdw,
Poland
Received October 20, 1987 Egg white lysozyme treated with hypochlorous acid links together producing di-, tri-, tetra-, and pentameric derivatives with molecular masses ranging from 14,300 to 90,500. Similar oligomeric products may be obtained by treating lysozyme color derivatives produced by labeling lysozyme with fluorescein, trinitrobenzenesulfonic acid and 2,4-dinitrofluorobenzene, with hypochlorous acid. The oligomeric lysozyme derivatives thus obtained consist ofa mixture of proteins with molecular massesequal to multiples of 14,300 (lysozyme molecular mass). This mixture can be applied as a set of molecular mass standards suitable for determination of protein molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0 1988 Academic
Press, Inc.
protein oxidation; lysozyme polymerization; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; molecular mass standards; low molecular weight proteins; protein labeling. KEY
WORDS:
Electrophoresis of proteins on polyacrylamide gel in a sodium dodecyl sulfate (SDS)’ solution has become one of most commonly used methods for the determination of protein molecular masses (I). However, this method requires using molecular mass standards, necessary for reliable calculation of molecular mass of the protein studied. For this purpose sets of specially selected natural proteins or mixtures of chemically crosslinked proteins with estimated molecular masses are commonly used. In this paper we describe a procedure for obtaining oligomers of the egg white lysozyme which can be employed as suitable molecular mass standards in the range of 14,300 to 90,500. MATERIALS
AND METHODS
Lysozyme of egg white, three times crystallized, dialyzed, and lyophilized; molecular mass markers MW-SDS-70 and MW-SDS’ Abbreviations used: SDS, sodium dodecyl sulfate; TNBSA, 2,4,6-trinitrobenzenesulfonic acid: DNBF, 2,4-dinitroflurobenzene. 419
280; fluorescein isothiocyanate; 2,4-dinitrofluorobenzene; and sodium dodecyl sulfate were purchased from Sigma Chemical Co. 2,4,6-Trinitrobenzenesulfonic acid, N,Nmethylene bisacrylamide, acrylamide, and amido black were from Serva Fine Chemicals. Tetramethylethylendiamine and sodium hypochlorite solution were obtained from BDH Chemicals Ltd. All other reagents were of analytical grade obtained from Poch Chemicals (Gliwice, Poland). Preparation of lysozyme oligomeric derivatives. To obtain lysozyme oligomeric derivatives, 10 mg of lysozyme was dissolved in 5 ml of 0.05 M citrate buffer, pH 4.5; then 8.7 mg of arginine was added to the solution up to a final arginine concentration of 0.01 M. Then 200 ~1 of 0.1 sodium hypochlorite was added to the prepared solution. A white insoluble deposit that precipitated in a few minutes was the reaction product. The sample was left at room temperature for exactly 10 min and then centrifuged for 5 min at 1OOOg.The supernatant was discarded and the precipitate was resuspended in 1 ml of distilled water and centrifuged again. The procedure of washing 0003-2697/88 Copyright
$3.00
0 1988 by Academic
Press, Inc.
420
DROiDi
AND
with water was repeated two more times. The sediment finally obtained was dissolved in 5 ml of 0.035 M sodium dodecyl sulfate solution in 0.01 M phosphate buffer, pH 7.2. Further addition of saccharose up to 0.3 M yielded a stable solution of the lysozyme oligomers mixture. This mixture could be stored frozen for several months without any detectable change in properties. Preparation ofjluorescein-labeled lysozyme oZigomers. Fluorescein-labeled lysozyme derivative was obtained using the procedure described by Clausen (2). This was performed as follows: 10 mg of lysozyme was dissolved in 5 ml of 0.1 M carbonate buffer, pH 9.2, and the solution was cooled to 10°C. Separately a solution of 2 mg of fluorescein isothiocyanate was added to the lysozyme solution upon vigorous stirring. The mixture was left for 18 h at 10°C and then the acidity of the mixture was adjusted to pH 4.5 by addition of 1 M citric acid. The oligomers of fluorescein-labeled lysozyme derivative were obtained as described above for native lysozyme. Preparation of 2,4,6-trinitrobenzenesulfonic acid-labeled lysozyme oligomers. Lysozyme labeling with 2,4,6-trinitrobenzenesulfonic acid (TNBSA) was performed by adding 15 mg of TNBSA to 5 ml of lysozyme solution 2 mg/ml in 0.05 M sodium carbonate buffer, pH 9.2. The mixture was stirred at room temperature for 1 h, and then the acidity of the solution was adjusted to pH 4.5 by addition of citric acid. Polymerization of color lysozyme-TNBSA derivative was obtained as described above. Preparation of 2,4-dinitrojluorobenzenelabeied Zysozyme polymers. Lysozyme coupling with 2,4-dinitrofluorobenzene (DNBF) was performed by adding 10 ~1 of DNBF to 5 ml of lysozyme solution 2 mg/ml in 0.05 h4 carbonate buffer, pH 9.2. The reaction mixture was left at room temperature for 18 h. Then the acidity of the solution was adjusted to pH 4.5 with 1 M citric acid. The oligomerit derivatives of color DNFB-labeled lysozyme were obtained as described above. Electrophoretic procedures for separation of lysozyme derivatives. Separation of oligo-
NASKALSKI
merit lysozyme derivatives was carried out using SDS electrophoresis on a 7% polyacrylamide gel as described by Lubega (3). A standard chamber and gel polymerized in glass tubes (6 X 120 mm in diameter) were used. The current of intensity of 20 mM was applied per tube. Samples ( 100-200 ~1) of the studied material with protein concentration ranging from 0.5 to 2.0 mg/ml were applied on each gel tube. A water solution of bromphenol blue was used as an indicator of protein migration rate. After termination of the electrophoresis polyacrylamide gel rods were removed from the tubes and separated proteins were stained with amido black dye (1 g/liter solution in acetic acid, 1.2 M/liter). The staining was performed at room temperature for 17 h. The excess dye was washed out from the gel rods using a water-acetic
Migration
distance
(mm)
FIG. 1. SDS electrophoresis pattern of separated lysozyme oligomeric derivatives obtained by treatment of lysozyme with hypochlorite at pH 4.5. The protein band located at 76.8 mm represents the lysozyme monomer; the further bands located at 64.0 to 42.6 mm represent di-, tri-, tetra-, and further lysozyme oligomers, respectively. Protein staining was performed using amido black dye.
LYSOZYME
MOLECULAR
421
MASS STANDARDS
TABLE 1 COMPARISON CALCULATED
OF VALUES OF LOGARITHMS OF STANDARD USING LYSOZME OLIGOMERS MIXTURE
PROTEINS’ MOLECULAR AS MOLECULAR MASS
MASSES-ASSUMED STANDARD PROTEIN
AND SET
Protein studied
Relative migration distance
Assumed molecular mass
Logarithm of assumed molecular mass
Calculated value” of logarithm of molecular mass
Obtained difference” between assumed and calculated log(M,) values
Lysozyme Hb (monomerp Trypsinogen Hb (dimer) Pepsin Albumin (egg) Hb (trimer) Hb (tetramer) Albumin (bovine)
0.8059 0.8 143 0.6297 0.5913 0.5290 0.4660 0.4390 0.35 10 0.3464
14,300 16,000 24,000 32,000 34,700 45,000 48,000 64,000 66,000
4.155 4.204 4.380 4.505 4.540 4.653 4.68 1 4.806 4.820
4.178 4.164 4.42 1 4.473 4.560 4.646 4.683 4.805 4.810
+0.55% -0.95% +0.94% -0.71% +0.44% -0.15% +0.0496 -0.02% -0.21%
’ Mean of three estimations. h Hb, Hemoglobin (Sigma).
acid-methanol solution in proportions 83:7:10 (v/v). Then the gel rods placed in glass cuvettes were scanned using an Autoradiogram-Gelscanner device and a Beckmann DU 8B UV/Vis Spectrophotometer. Gel optical density values were plotted versus length of the scanned gel. Calculation of protein molecular masses was performed em-
I
0
.I
.2 Relative
.3
.4
.s
migration
.6 distance
7
.8
.8
Rf
FIG. 2. Logarithms of the molecular mass values plotted versus relative migration distances (&values) of lysozyme oligomers and other standard proteins with estimated molecular masses.
ploying a Beckmann Gel-Scan Compuset III molecular mass determination program (4). RESULTS AND DISCUSSION
Lysozyme treated with hypochlorous acid polymerizes giving as reaction product a set of protein bands located in the gel rods in positions corresponding to multiples of 14,300 (Fig. 1). In all experiments, when lysozyme oligomers were obtained, at least five sharp protein bands, in addition to the monomeric lysozyme band and corresponding to di-, tri-, tetra-, and pentameric derivatives, were always observed. Assuming that the molecular mass of the derivatives obtained was a multiple of 14,300, a perfect linear relationship between the logarithm of the assumed molecular mass values and the respective migration distance in SDS-polyacrylamide gel electrophoresis was observed (Fig. 2). To check the usefulness of lysozyme-polymerized derivatives as molecular mass standards, we used them to determine molecular masses of selected proteins. For this purpose a SDS electrophoresis molecular mass standard proteins set supplied by Sigma-London was used. As shown in Table 1, the logarithms of molecular masses of the analyzed
422
DROiDi
20
I
100
70
Migration
distance
AND NASKALSKI
(mm)
FIG. 3. Densitogram of lysozyme oligomers labeled with trinitrobenzenesulfonic acid and separated on a SDS-polyacrylamide gel. The gel rod was scanned at light 420 nm directly after termination of the electrophoresis without any further protein staining.
proteins did not differ more than 1% from their anticipated values. The use of lysozyme oligomer preparations stored in a freezer for 2 years did not show any difference in results of the performed determinations. Lysozyme polymeric derivatives linked with chromophoric residues yielded separation patterns the same as those of colorless lysozyme polymers. The color of the migrating proteins made it possible to follow directly the migration process, making the staining of proteins after the termination of electrophoresis unnecessary. Scanning the labeled lysozyme derivatives yielded migration patterns the same as those obtained for the unlabeled lysozyme (Fig. 3). Polymerization of lysozyme was first observed as a final stage of lysozyme oxidation with myeloperoxidase, Ci-, and a hydrogen peroxide system (5). The mechanism of the crosslinking of lysozyme molecules remains
unclear, but the products obtained are stable under @-mercaptoethanol treatment and do not dissociate in SDS solutions. Thus the lysozyme oligomers constitute a set of stable proteins with precisely known molecular masses, and are therefore suitable for use in SDS electrophoresis. As shown in this paper, the preparation and use of lysozyme oligomers are simple enough to be performed in any laboratory. The peculiarity of lysozyme oligomers when used as molecular mass standards is that each standard differs from the other one by a constant, well-defined value. This produces a uniquely uniform pattern of protein bands in SDS electrophoretic pictures, enabling precise calculation of data for equations used for further estimations of molecular masses of proteins studied. In our experiments when using different sets of heterogenous natural proteins as standards the egg white albumin molecular mass determined on SDS-polyacrylamide gel electrophoresis differed by up to 10%. On the other hand when using the lysozyme oligomers as standards the bias was about 1% only. Apart from uniformity, the lysozyme oligomers are considerably cheaper than any other molecular mass standard set commercially available. Therefore it seems that the described procedure of obtaining and use of lysozyme derivatives is of some practical value for determining of protein molecular masses. REFERENCES 1. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 244,4406-44 12. 2. Clausen, J. (1969) Laboratory Techniques in Biochemistry and Molecular Biology: Immunochemical Techniques for the Identification and Estimation of Macromolecules. North-Holland, Amsterdam/London. 3. Lubega, J. (1983) Clin. Chim. Acta 128, 151-167. 4. Beckman Instruments (1980) Beckman DU 8 Gel Scanning System: A Preliminary Instruction Manual, pp. 34-54, Beckman Instruments, Inc., Scientific Instruments Div., Fullerton, CA, 5. Droidi, R., et al. (1988) Acta B&him. Pal. 35, in press.
171,419-422
BIOCHEMISTRY
Lysozyme
(1988)
Oligomers as a Molecular Sulfate-Polyacrylamide
Mass Standard for Sodium Dodecyl Gel Electrophoresis
RYSZARD DRO~D~ AND JERZY W. NASKALSKI Department
of Biochemical
Diagnostics,
Medical
Academy,
Krakdw,
Poland
Received October 20, 1987 Egg white lysozyme treated with hypochlorous acid links together producing di-, tri-, tetra-, and pentameric derivatives with molecular masses ranging from 14,300 to 90,500. Similar oligomeric products may be obtained by treating lysozyme color derivatives produced by labeling lysozyme with fluorescein, trinitrobenzenesulfonic acid and 2,4-dinitrofluorobenzene, with hypochlorous acid. The oligomeric lysozyme derivatives thus obtained consist ofa mixture of proteins with molecular massesequal to multiples of 14,300 (lysozyme molecular mass). This mixture can be applied as a set of molecular mass standards suitable for determination of protein molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0 1988 Academic
Press, Inc.
protein oxidation; lysozyme polymerization; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; molecular mass standards; low molecular weight proteins; protein labeling. KEY
WORDS:
Electrophoresis of proteins on polyacrylamide gel in a sodium dodecyl sulfate (SDS)’ solution has become one of most commonly used methods for the determination of protein molecular masses (I). However, this method requires using molecular mass standards, necessary for reliable calculation of molecular mass of the protein studied. For this purpose sets of specially selected natural proteins or mixtures of chemically crosslinked proteins with estimated molecular masses are commonly used. In this paper we describe a procedure for obtaining oligomers of the egg white lysozyme which can be employed as suitable molecular mass standards in the range of 14,300 to 90,500. MATERIALS
AND METHODS
Lysozyme of egg white, three times crystallized, dialyzed, and lyophilized; molecular mass markers MW-SDS-70 and MW-SDS’ Abbreviations used: SDS, sodium dodecyl sulfate; TNBSA, 2,4,6-trinitrobenzenesulfonic acid: DNBF, 2,4-dinitroflurobenzene. 419
280; fluorescein isothiocyanate; 2,4-dinitrofluorobenzene; and sodium dodecyl sulfate were purchased from Sigma Chemical Co. 2,4,6-Trinitrobenzenesulfonic acid, N,Nmethylene bisacrylamide, acrylamide, and amido black were from Serva Fine Chemicals. Tetramethylethylendiamine and sodium hypochlorite solution were obtained from BDH Chemicals Ltd. All other reagents were of analytical grade obtained from Poch Chemicals (Gliwice, Poland). Preparation of lysozyme oligomeric derivatives. To obtain lysozyme oligomeric derivatives, 10 mg of lysozyme was dissolved in 5 ml of 0.05 M citrate buffer, pH 4.5; then 8.7 mg of arginine was added to the solution up to a final arginine concentration of 0.01 M. Then 200 ~1 of 0.1 sodium hypochlorite was added to the prepared solution. A white insoluble deposit that precipitated in a few minutes was the reaction product. The sample was left at room temperature for exactly 10 min and then centrifuged for 5 min at 1OOOg.The supernatant was discarded and the precipitate was resuspended in 1 ml of distilled water and centrifuged again. The procedure of washing 0003-2697/88 Copyright
$3.00
0 1988 by Academic
Press, Inc.
420
DROiDi
AND
with water was repeated two more times. The sediment finally obtained was dissolved in 5 ml of 0.035 M sodium dodecyl sulfate solution in 0.01 M phosphate buffer, pH 7.2. Further addition of saccharose up to 0.3 M yielded a stable solution of the lysozyme oligomers mixture. This mixture could be stored frozen for several months without any detectable change in properties. Preparation ofjluorescein-labeled lysozyme oZigomers. Fluorescein-labeled lysozyme derivative was obtained using the procedure described by Clausen (2). This was performed as follows: 10 mg of lysozyme was dissolved in 5 ml of 0.1 M carbonate buffer, pH 9.2, and the solution was cooled to 10°C. Separately a solution of 2 mg of fluorescein isothiocyanate was added to the lysozyme solution upon vigorous stirring. The mixture was left for 18 h at 10°C and then the acidity of the mixture was adjusted to pH 4.5 by addition of 1 M citric acid. The oligomers of fluorescein-labeled lysozyme derivative were obtained as described above for native lysozyme. Preparation of 2,4,6-trinitrobenzenesulfonic acid-labeled lysozyme oligomers. Lysozyme labeling with 2,4,6-trinitrobenzenesulfonic acid (TNBSA) was performed by adding 15 mg of TNBSA to 5 ml of lysozyme solution 2 mg/ml in 0.05 M sodium carbonate buffer, pH 9.2. The mixture was stirred at room temperature for 1 h, and then the acidity of the solution was adjusted to pH 4.5 by addition of citric acid. Polymerization of color lysozyme-TNBSA derivative was obtained as described above. Preparation of 2,4-dinitrojluorobenzenelabeied Zysozyme polymers. Lysozyme coupling with 2,4-dinitrofluorobenzene (DNBF) was performed by adding 10 ~1 of DNBF to 5 ml of lysozyme solution 2 mg/ml in 0.05 h4 carbonate buffer, pH 9.2. The reaction mixture was left at room temperature for 18 h. Then the acidity of the solution was adjusted to pH 4.5 with 1 M citric acid. The oligomerit derivatives of color DNFB-labeled lysozyme were obtained as described above. Electrophoretic procedures for separation of lysozyme derivatives. Separation of oligo-
NASKALSKI
merit lysozyme derivatives was carried out using SDS electrophoresis on a 7% polyacrylamide gel as described by Lubega (3). A standard chamber and gel polymerized in glass tubes (6 X 120 mm in diameter) were used. The current of intensity of 20 mM was applied per tube. Samples ( 100-200 ~1) of the studied material with protein concentration ranging from 0.5 to 2.0 mg/ml were applied on each gel tube. A water solution of bromphenol blue was used as an indicator of protein migration rate. After termination of the electrophoresis polyacrylamide gel rods were removed from the tubes and separated proteins were stained with amido black dye (1 g/liter solution in acetic acid, 1.2 M/liter). The staining was performed at room temperature for 17 h. The excess dye was washed out from the gel rods using a water-acetic
Migration
distance
(mm)
FIG. 1. SDS electrophoresis pattern of separated lysozyme oligomeric derivatives obtained by treatment of lysozyme with hypochlorite at pH 4.5. The protein band located at 76.8 mm represents the lysozyme monomer; the further bands located at 64.0 to 42.6 mm represent di-, tri-, tetra-, and further lysozyme oligomers, respectively. Protein staining was performed using amido black dye.
LYSOZYME
MOLECULAR
421
MASS STANDARDS
TABLE 1 COMPARISON CALCULATED
OF VALUES OF LOGARITHMS OF STANDARD USING LYSOZME OLIGOMERS MIXTURE
PROTEINS’ MOLECULAR AS MOLECULAR MASS
MASSES-ASSUMED STANDARD PROTEIN
AND SET
Protein studied
Relative migration distance
Assumed molecular mass
Logarithm of assumed molecular mass
Calculated value” of logarithm of molecular mass
Obtained difference” between assumed and calculated log(M,) values
Lysozyme Hb (monomerp Trypsinogen Hb (dimer) Pepsin Albumin (egg) Hb (trimer) Hb (tetramer) Albumin (bovine)
0.8059 0.8 143 0.6297 0.5913 0.5290 0.4660 0.4390 0.35 10 0.3464
14,300 16,000 24,000 32,000 34,700 45,000 48,000 64,000 66,000
4.155 4.204 4.380 4.505 4.540 4.653 4.68 1 4.806 4.820
4.178 4.164 4.42 1 4.473 4.560 4.646 4.683 4.805 4.810
+0.55% -0.95% +0.94% -0.71% +0.44% -0.15% +0.0496 -0.02% -0.21%
’ Mean of three estimations. h Hb, Hemoglobin (Sigma).
acid-methanol solution in proportions 83:7:10 (v/v). Then the gel rods placed in glass cuvettes were scanned using an Autoradiogram-Gelscanner device and a Beckmann DU 8B UV/Vis Spectrophotometer. Gel optical density values were plotted versus length of the scanned gel. Calculation of protein molecular masses was performed em-
I
0
.I
.2 Relative
.3
.4
.s
migration
.6 distance
7
.8
.8
Rf
FIG. 2. Logarithms of the molecular mass values plotted versus relative migration distances (&values) of lysozyme oligomers and other standard proteins with estimated molecular masses.
ploying a Beckmann Gel-Scan Compuset III molecular mass determination program (4). RESULTS AND DISCUSSION
Lysozyme treated with hypochlorous acid polymerizes giving as reaction product a set of protein bands located in the gel rods in positions corresponding to multiples of 14,300 (Fig. 1). In all experiments, when lysozyme oligomers were obtained, at least five sharp protein bands, in addition to the monomeric lysozyme band and corresponding to di-, tri-, tetra-, and pentameric derivatives, were always observed. Assuming that the molecular mass of the derivatives obtained was a multiple of 14,300, a perfect linear relationship between the logarithm of the assumed molecular mass values and the respective migration distance in SDS-polyacrylamide gel electrophoresis was observed (Fig. 2). To check the usefulness of lysozyme-polymerized derivatives as molecular mass standards, we used them to determine molecular masses of selected proteins. For this purpose a SDS electrophoresis molecular mass standard proteins set supplied by Sigma-London was used. As shown in Table 1, the logarithms of molecular masses of the analyzed
422
DROiDi
20
I
100
70
Migration
distance
AND NASKALSKI
(mm)
FIG. 3. Densitogram of lysozyme oligomers labeled with trinitrobenzenesulfonic acid and separated on a SDS-polyacrylamide gel. The gel rod was scanned at light 420 nm directly after termination of the electrophoresis without any further protein staining.
proteins did not differ more than 1% from their anticipated values. The use of lysozyme oligomer preparations stored in a freezer for 2 years did not show any difference in results of the performed determinations. Lysozyme polymeric derivatives linked with chromophoric residues yielded separation patterns the same as those of colorless lysozyme polymers. The color of the migrating proteins made it possible to follow directly the migration process, making the staining of proteins after the termination of electrophoresis unnecessary. Scanning the labeled lysozyme derivatives yielded migration patterns the same as those obtained for the unlabeled lysozyme (Fig. 3). Polymerization of lysozyme was first observed as a final stage of lysozyme oxidation with myeloperoxidase, Ci-, and a hydrogen peroxide system (5). The mechanism of the crosslinking of lysozyme molecules remains
unclear, but the products obtained are stable under @-mercaptoethanol treatment and do not dissociate in SDS solutions. Thus the lysozyme oligomers constitute a set of stable proteins with precisely known molecular masses, and are therefore suitable for use in SDS electrophoresis. As shown in this paper, the preparation and use of lysozyme oligomers are simple enough to be performed in any laboratory. The peculiarity of lysozyme oligomers when used as molecular mass standards is that each standard differs from the other one by a constant, well-defined value. This produces a uniquely uniform pattern of protein bands in SDS electrophoretic pictures, enabling precise calculation of data for equations used for further estimations of molecular masses of proteins studied. In our experiments when using different sets of heterogenous natural proteins as standards the egg white albumin molecular mass determined on SDS-polyacrylamide gel electrophoresis differed by up to 10%. On the other hand when using the lysozyme oligomers as standards the bias was about 1% only. Apart from uniformity, the lysozyme oligomers are considerably cheaper than any other molecular mass standard set commercially available. Therefore it seems that the described procedure of obtaining and use of lysozyme derivatives is of some practical value for determining of protein molecular masses. REFERENCES 1. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 244,4406-44 12. 2. Clausen, J. (1969) Laboratory Techniques in Biochemistry and Molecular Biology: Immunochemical Techniques for the Identification and Estimation of Macromolecules. North-Holland, Amsterdam/London. 3. Lubega, J. (1983) Clin. Chim. Acta 128, 151-167. 4. Beckman Instruments (1980) Beckman DU 8 Gel Scanning System: A Preliminary Instruction Manual, pp. 34-54, Beckman Instruments, Inc., Scientific Instruments Div., Fullerton, CA, 5. Droidi, R., et al. (1988) Acta B&him. Pal. 35, in press.