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Electrophoretic characterization of plasma RNA
BIOCHEMICAL
MEDICINE
13, 224-230 (1975)
Electrophoretic
Characterization
of Plasma
RNA
LARRY W. GUIN, K. E. GRISWOLD,~ SELMA PATTON, RICHARD C. KAMM, AND ALBERT G. SMITH Department of Pathology, Louisiana State University Medical Center, School of Medicine in Shreveport, P. 0. Box 3932, Shreveport, Louisiana 71130 Received April 21, 1975
Several studies of the quantity of total ribonucleic acid (RNA) in whole blood have been made (l-5). Recently the studies have been extended to determine the quantity of total ribonucleic acid in plasma (6,7). An average of 144 k’22 mg of ribonucleic acid per liter of plasma has been reported in a study of 286 healthy subjects using a rapid inexpensive fluorometric procedure (7). The method has recently been automated by Elzen and Kamm (8). Many RNA types have been quantitated and identified on the basis of molecular weight, sedimentation coefficient, and electrophoretic motility (9-l 1). Peacock and Dingman (12) fractionated RNA extracted from rat tissue using polyacrylamide-agarose composite gels. They were able to characterize the RNAs present without prior purification by comparing their migration relative to RNA of known molecular weight. Since there is an exchange of certain materials between tissues and vascular system, plasma RNA may be as heterogenous as cellular RNA. The purpose of this study is to utilize the technique of Kamm and Smith (7) for the quantitation of total plasma RNA and combine this with an application of the techniques of Peacock and Dingman (9) and Dingman and Peacock (13) for the fractionation of tissue RNA by making those modifications necessary to adapt it for the quantitation of electrophoretic fractions of plasma RNA. MATERIALS
AND
METHODS
Blood samples were obtained from apparently healthy individuals of both sexes, ages 3 to 62, who had no major illnesses during the last 2 years. The specimens were collected and immediately mixed with sodium EDTA. Equal amounts of whole blood and a ribonuclease inhibi’ Reprint requests to K. E. Griswold, Ph.D. 224 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
ELECTROPHORESIS
OF
PLASMA
RNA
225
tor, iodoacetic acid, were mixed according to the method of Kamm (6) and Kamm and Smith (7). The inhibited sample was mixed, centrifuged, the plasma separated, and divided into two aliquots. One aliquot of the inhibited plasma was used to determine the total plasma RNA concentration according to the method of Kamm and Smith (7) using the modification of Elzen and Kamm (8), and the other aliquot was used to determine RNA fractions by electrophoresis. To each aliquot 0.5 mg of deoxyribonuclease (100 ~1 of 5 mg/ml solution) was added to eliminate the deoxyribonucleic acid. Electrophoresis was performed in a vertical electrophoretic cell, model 470, using a model 454 power supply (E. C. Apparatus Company, 3831 Tyrone Boulevard, North, St. Petersburg, Florida). A stock Tris-EDTA buffer (pH 8.3) was used in the preparation of a 3.5% polyacrylamide-agarose gel according to the method of Dingman and Peacock (13). The gel was poured into the cell and allowed to polymerize. The cell was then placed in a vertical position and both chambers filled with reservoir buffer (stock buffer diluted 1:lO with deionized water). An EC-142 buffer pump was used to circulate ice water (4-6”) through the cooling plates throughout the procedure, but reservoir buffer was not recirculated. The power supply was then connected to the cell, and ionic equilibrium in the gel effected by a 45minute prerun at 200 V. After the prernn, the power supply was disconnected and the buffer in the upper compartment was lowered to a level just below the gel slot. Buffer was removed from the sample slot to facilitate sample application and help prevent sample mixing. A 300-k] sample of the inhibited plasma was applied to the gel sample slot in duplicate. A 100-p] sample of Escherichia coli 4-S transfer RNA (10 pug/100 ~1 in 5% sucrose solution) was used as a reference. The buffer level in the upper compartment was maintained at its original level, the power supply reconnected, and electrophoresis proceeded for 1.5 hours at 200 V. After fractionation the gel slab was removed from the cell, rinsed for 20-30 minutes in 1 M acetic acid to drop the pH of the gel and stained by the toluidine blue 0 technique of Peacock and Dingman (9) using a 12- to 16-hour staining period which was the staining period required for staining of RNA fractions in these 6-mm thick gels. Destaining was accomplished by successive changes of distilled water over a 2- to 3-day period. Other staining procedures, including pyronine Y, acridine orange, methylene blue, Azure A, and ethidium bromide, were evaluated, but the toluidine blue 0 technique was selected as the technique which gave the most intensely stained fractions. Each electropherogram was scanned densitometrically at 610 nm and the percentage of total plasma RNA present in each band calculated.
226
GUIN
ET AL.
RESULTS In the electrophoretic fractionation of plasma RNA from 51 apparently healthy individuals a total of five bands of RNA activity was distributed in three distinct banding patterns (see Fig. 1). All RNA bands migrated in the range of 6-27 S RNA relative to the migration of E. coli 4s tRNA. The mean total RNA concentration for this group was 144 +-‘35 mgihter of plasma. In all specimens, two predominate bands, 3 and 4, were present, in combination with either a single broad, diffuse band, 1 (Pattern A); or
A
B
C
D
FIG. 1. Photograph of the three electrophoretic banding patterns, which shows the number of each band, and the reference sample. From left to right: slot 1, pattern A; slot 2, pattern B; slot 3, pattern C; slot 4, Escherichia coli 4 S tRNA.
ELECTROPHORESIS
OF PLASMA
227
RNA
I
Origin (-)
* (4
FIG. 2. Densitometric readings of the three banding patterns. From top to bottom: I. pattern A: II, pattern B; III, pattern C.
two more discrete bands, 1 and 2 (Pattern B). Plasma from 2 of the 51 individuals gave a third pattern (Pattern C), which consisted of bands 1, 3, 4, and another band (band 5), which migrated anodic to band 4. A densitometric composite of the three patterns are seen in Fig. 2. Table 1 gives the mean total plasma RNA concentration and mean percentage of total concentration for each band in each of the three banding patterns.
TABLE TOTAL
RNA CONCENTRATION PLASMA
1
AND MEAN % FOR RNA ELECTROPHORETIC PATTERNS
BANDS
FOUND
IN
Mean % of total RNA for each band Pattern A (n = 39) SD B (n = 10) SD C(fl= 2) SD
Mean total RNA concentration (mg/liter) 142.8 38.8 141.0 34.7 182.5 31.8
1
35.2 7.7 19.7 6.1 31.1 2.5
2
3
4
5
22.0 6.7
37.8 7.3 32.6 5.2 33.6 0.7
27.0 4.7 26.0 4.7 16.6 4.7
18.7 2.8
228
GUIN
ET
AL.
DISCUSSION
Fractionation of plasma RNA on polyacrylamide-agarose gels revealed very similar, yet distinct banding patterns among normal individuals. This similarity in electropherograms of plasma RNA reflects the uniformity of RNA banding patterns observed in a variety of tissue RNAs by Peacock and Dingman (9). Since there is an exchange of many macromolecules between the intra- and extracellular compartments the study of plasma RNA could be a reflection of RNA molecular constitution in intracellular and pericellular areas. In this study, all plasma RNA bands migrated in a range of IO-32 mm under conditions described by Peacock and Dingman (12). Reference E. coli 4s tRNA migrated a distance of 70 mm under the same conditions. Peacock and Dingman (12) reported the following migratory rates for liver RNA: 3 mm for 30 S RNA; 13-14 mm for 18 S doublet RNA: 60 mm for 5 S RNA; and 67 mm for 4 S RNA. Under the same conditions, E. coli RNA migrated as follows: 7 mm for 23 S RNA; 15-16 mm for 16 S doublet RNA; 62 mm for 5 S RNA; and 67 mm for 4 S RNA. Using these reference points, the observed plasma RNA bands migrated in the range of 6-27 S RNA and no low molecular weight RNA was detected. In animal cells, r-RNA consists of 28, 18, and 5 S material (14); m-RNA, of 6-25 S material and t-RNA, of about 4 S material (15). These studies show that plasma RNA could be m-RNA or r-RNA, but not t-RNA. In all banding patterns, band 3 appears in a relatively uniform percentage of concentration. In patterns A and B, band 4 is also found in a similarly uniform percentage of concentration., Though band 4 was observed in Pattern C, the percentage of concentration was much lower than observed in the other groups. In addition to having a lower mean percentage of concentration of band 4, Pattern C also exhibited a unique, electrophoretically distinct, band 5, which migrated slightly anodic to band 4. This pattern was observed in only two young females, ages 21 and 23. Band 1 appeared as a broad diffuse band in Patterns A and C, comprising an average of 35.2% of the total RNA in the former and 3 1.1% in the latter. The band was narrower in Pattern B with an average concentration of 19.7% of the total RNA. Band 2 comprised an average of 22.0% of the total RNA in 10 individuals and could be delineated densitometrically from the broad, band 1, noted in Patterns A and C (see Fig. 2.) Peacock and Dingman (9) found that deliberately degraded specimens of RNA contained broad, highly dispersed zones of RNA-staining material. The diffuseness noted in bands 1 and 2 and/or the resolution of a fifth band in some specimens may result from similar in vivo or in vitro degradation. Alternately, inconsistancies in bands 1 and 2 and/or 5 may
ELECTROPHORESIS
OF PLASMA
RNA
229
be attributed to the heterogeneity known to exist in RNA molecules. Such heterogeneity has been observed in ionograms of tissue RNA obtained from all major sedimentation zones (9, 12, 13). It is important to note that when whole cytoplasmic RNA is fractionated electrophoretically, the same minor components are present which are noted in electropherograms of fractions after sucrose gradient separation, that patterns are highly reproducible from preparation to preparation, and from tissue to tissue in the rat (9). Though the mean total plasma RNA concentration of 144 mg/liter obtained for the population in this study agreed closely with that reported for a healthy population by Kamm and Smith (7), a considerable range in total concentration was found (50-215 mg/liter) among individuals in the present study. This wide range in total RNA concentration and the variation-in electrophoretic patterns may be due to differences in the metabolic condition of the individual. Broader and more detailed electrophoretie characterizations of plasma RNA are needed to provide additional information that could be beneficial in monitoring the health state of an individual. SUMMARY
Electrophoresis on polyacrylamide-agarose composite gels was used to fractionate plasma RNA from 51 normal individuals. The RNA fractions all migrated as higher molecular weights species in the range of 6-27 S RNA when compared to an Escherichia coli 4 S tRNA reference. In all cases two predominate bands, 3 and 4, were present; with either a single broad diffuse band 1, or two more discrete bands, 1 and 2. Two out of 51 individuals had a fifth more anodic band than band 4. Approximately 76% of the individuals surveyed had an identical banding pattern. Two variants of this pattern comprised 20 and 4% of the total surveyed. Only two females, ages 21 and 23, exhibited the fifth band. No other apparent differences were noted among age and sex groups, nor could any correlation between pattern variants and total plasma RNA concentrations be detected. REFERENCES 1. Javillier, M. and Fabrykant, M., Bull. Sot. Chim. Biol. 13, 1253 (1931). 2. Mandel, P. and Metais, P., C. R. Acad. Sci. 112, 16 (1947). 3. Niemer, H. and Sadtler, E., Klin. Wochenschr. 27, 278 (1949). 4. Fleck, A. and Beg, D., Biochim. Biophys. Acta 42, 333 (1966). 5. Kaznacheev, V. P. and Polyakov, Y. V., Kardiologiga 7, 22 (1967). 6. Kamm, R. C., Dissertation. University of Tennessee, 1970. 7. Kamm, R. C. and Smith, A. G., Clin. Chem. 18, 51%522(1972). 8. Elzen, G. and Kamm, R. C., Camp. Rio&em. Physiol. 4Sa, $81 (1974). 9. Peacock, A. C. and Dingman, C. W., Biochemistry 6, 1818 (1967). 10. McConkey, E. H. and Hopkins, J. W., J. Mol. Biol. 39, 54.5 (1969).
230
GUIN
11. 12. 13. 14.
ET AL.
Granboulan, N. and Scherrer, K., Eur. J. Biochem. 9, 1 (1969). Peacock, A. C. and Dingman, C. W., Biochemistry 7, 668 (1968). Dingman, C. W. and Peacock, A. C., Biochemisrry 7, 659 (1968). Orten, J. M. and Neuhaus, D. W., “Biochemistry.” C. V. Mosby Co.. St. Louis, Missouri, 1970. 15. Lehninger, A. L., “Biochemistry.” Worth Publishers, Inc., New York, New York 197-o.
MEDICINE
13, 224-230 (1975)
Electrophoretic
Characterization
of Plasma
RNA
LARRY W. GUIN, K. E. GRISWOLD,~ SELMA PATTON, RICHARD C. KAMM, AND ALBERT G. SMITH Department of Pathology, Louisiana State University Medical Center, School of Medicine in Shreveport, P. 0. Box 3932, Shreveport, Louisiana 71130 Received April 21, 1975
Several studies of the quantity of total ribonucleic acid (RNA) in whole blood have been made (l-5). Recently the studies have been extended to determine the quantity of total ribonucleic acid in plasma (6,7). An average of 144 k’22 mg of ribonucleic acid per liter of plasma has been reported in a study of 286 healthy subjects using a rapid inexpensive fluorometric procedure (7). The method has recently been automated by Elzen and Kamm (8). Many RNA types have been quantitated and identified on the basis of molecular weight, sedimentation coefficient, and electrophoretic motility (9-l 1). Peacock and Dingman (12) fractionated RNA extracted from rat tissue using polyacrylamide-agarose composite gels. They were able to characterize the RNAs present without prior purification by comparing their migration relative to RNA of known molecular weight. Since there is an exchange of certain materials between tissues and vascular system, plasma RNA may be as heterogenous as cellular RNA. The purpose of this study is to utilize the technique of Kamm and Smith (7) for the quantitation of total plasma RNA and combine this with an application of the techniques of Peacock and Dingman (9) and Dingman and Peacock (13) for the fractionation of tissue RNA by making those modifications necessary to adapt it for the quantitation of electrophoretic fractions of plasma RNA. MATERIALS
AND
METHODS
Blood samples were obtained from apparently healthy individuals of both sexes, ages 3 to 62, who had no major illnesses during the last 2 years. The specimens were collected and immediately mixed with sodium EDTA. Equal amounts of whole blood and a ribonuclease inhibi’ Reprint requests to K. E. Griswold, Ph.D. 224 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
ELECTROPHORESIS
OF
PLASMA
RNA
225
tor, iodoacetic acid, were mixed according to the method of Kamm (6) and Kamm and Smith (7). The inhibited sample was mixed, centrifuged, the plasma separated, and divided into two aliquots. One aliquot of the inhibited plasma was used to determine the total plasma RNA concentration according to the method of Kamm and Smith (7) using the modification of Elzen and Kamm (8), and the other aliquot was used to determine RNA fractions by electrophoresis. To each aliquot 0.5 mg of deoxyribonuclease (100 ~1 of 5 mg/ml solution) was added to eliminate the deoxyribonucleic acid. Electrophoresis was performed in a vertical electrophoretic cell, model 470, using a model 454 power supply (E. C. Apparatus Company, 3831 Tyrone Boulevard, North, St. Petersburg, Florida). A stock Tris-EDTA buffer (pH 8.3) was used in the preparation of a 3.5% polyacrylamide-agarose gel according to the method of Dingman and Peacock (13). The gel was poured into the cell and allowed to polymerize. The cell was then placed in a vertical position and both chambers filled with reservoir buffer (stock buffer diluted 1:lO with deionized water). An EC-142 buffer pump was used to circulate ice water (4-6”) through the cooling plates throughout the procedure, but reservoir buffer was not recirculated. The power supply was then connected to the cell, and ionic equilibrium in the gel effected by a 45minute prerun at 200 V. After the prernn, the power supply was disconnected and the buffer in the upper compartment was lowered to a level just below the gel slot. Buffer was removed from the sample slot to facilitate sample application and help prevent sample mixing. A 300-k] sample of the inhibited plasma was applied to the gel sample slot in duplicate. A 100-p] sample of Escherichia coli 4-S transfer RNA (10 pug/100 ~1 in 5% sucrose solution) was used as a reference. The buffer level in the upper compartment was maintained at its original level, the power supply reconnected, and electrophoresis proceeded for 1.5 hours at 200 V. After fractionation the gel slab was removed from the cell, rinsed for 20-30 minutes in 1 M acetic acid to drop the pH of the gel and stained by the toluidine blue 0 technique of Peacock and Dingman (9) using a 12- to 16-hour staining period which was the staining period required for staining of RNA fractions in these 6-mm thick gels. Destaining was accomplished by successive changes of distilled water over a 2- to 3-day period. Other staining procedures, including pyronine Y, acridine orange, methylene blue, Azure A, and ethidium bromide, were evaluated, but the toluidine blue 0 technique was selected as the technique which gave the most intensely stained fractions. Each electropherogram was scanned densitometrically at 610 nm and the percentage of total plasma RNA present in each band calculated.
226
GUIN
ET AL.
RESULTS In the electrophoretic fractionation of plasma RNA from 51 apparently healthy individuals a total of five bands of RNA activity was distributed in three distinct banding patterns (see Fig. 1). All RNA bands migrated in the range of 6-27 S RNA relative to the migration of E. coli 4s tRNA. The mean total RNA concentration for this group was 144 +-‘35 mgihter of plasma. In all specimens, two predominate bands, 3 and 4, were present, in combination with either a single broad, diffuse band, 1 (Pattern A); or
A
B
C
D
FIG. 1. Photograph of the three electrophoretic banding patterns, which shows the number of each band, and the reference sample. From left to right: slot 1, pattern A; slot 2, pattern B; slot 3, pattern C; slot 4, Escherichia coli 4 S tRNA.
ELECTROPHORESIS
OF PLASMA
227
RNA
I
Origin (-)
* (4
FIG. 2. Densitometric readings of the three banding patterns. From top to bottom: I. pattern A: II, pattern B; III, pattern C.
two more discrete bands, 1 and 2 (Pattern B). Plasma from 2 of the 51 individuals gave a third pattern (Pattern C), which consisted of bands 1, 3, 4, and another band (band 5), which migrated anodic to band 4. A densitometric composite of the three patterns are seen in Fig. 2. Table 1 gives the mean total plasma RNA concentration and mean percentage of total concentration for each band in each of the three banding patterns.
TABLE TOTAL
RNA CONCENTRATION PLASMA
1
AND MEAN % FOR RNA ELECTROPHORETIC PATTERNS
BANDS
FOUND
IN
Mean % of total RNA for each band Pattern A (n = 39) SD B (n = 10) SD C(fl= 2) SD
Mean total RNA concentration (mg/liter) 142.8 38.8 141.0 34.7 182.5 31.8
1
35.2 7.7 19.7 6.1 31.1 2.5
2
3
4
5
22.0 6.7
37.8 7.3 32.6 5.2 33.6 0.7
27.0 4.7 26.0 4.7 16.6 4.7
18.7 2.8
228
GUIN
ET
AL.
DISCUSSION
Fractionation of plasma RNA on polyacrylamide-agarose gels revealed very similar, yet distinct banding patterns among normal individuals. This similarity in electropherograms of plasma RNA reflects the uniformity of RNA banding patterns observed in a variety of tissue RNAs by Peacock and Dingman (9). Since there is an exchange of many macromolecules between the intra- and extracellular compartments the study of plasma RNA could be a reflection of RNA molecular constitution in intracellular and pericellular areas. In this study, all plasma RNA bands migrated in a range of IO-32 mm under conditions described by Peacock and Dingman (12). Reference E. coli 4s tRNA migrated a distance of 70 mm under the same conditions. Peacock and Dingman (12) reported the following migratory rates for liver RNA: 3 mm for 30 S RNA; 13-14 mm for 18 S doublet RNA: 60 mm for 5 S RNA; and 67 mm for 4 S RNA. Under the same conditions, E. coli RNA migrated as follows: 7 mm for 23 S RNA; 15-16 mm for 16 S doublet RNA; 62 mm for 5 S RNA; and 67 mm for 4 S RNA. Using these reference points, the observed plasma RNA bands migrated in the range of 6-27 S RNA and no low molecular weight RNA was detected. In animal cells, r-RNA consists of 28, 18, and 5 S material (14); m-RNA, of 6-25 S material and t-RNA, of about 4 S material (15). These studies show that plasma RNA could be m-RNA or r-RNA, but not t-RNA. In all banding patterns, band 3 appears in a relatively uniform percentage of concentration. In patterns A and B, band 4 is also found in a similarly uniform percentage of concentration., Though band 4 was observed in Pattern C, the percentage of concentration was much lower than observed in the other groups. In addition to having a lower mean percentage of concentration of band 4, Pattern C also exhibited a unique, electrophoretically distinct, band 5, which migrated slightly anodic to band 4. This pattern was observed in only two young females, ages 21 and 23. Band 1 appeared as a broad diffuse band in Patterns A and C, comprising an average of 35.2% of the total RNA in the former and 3 1.1% in the latter. The band was narrower in Pattern B with an average concentration of 19.7% of the total RNA. Band 2 comprised an average of 22.0% of the total RNA in 10 individuals and could be delineated densitometrically from the broad, band 1, noted in Patterns A and C (see Fig. 2.) Peacock and Dingman (9) found that deliberately degraded specimens of RNA contained broad, highly dispersed zones of RNA-staining material. The diffuseness noted in bands 1 and 2 and/or the resolution of a fifth band in some specimens may result from similar in vivo or in vitro degradation. Alternately, inconsistancies in bands 1 and 2 and/or 5 may
ELECTROPHORESIS
OF PLASMA
RNA
229
be attributed to the heterogeneity known to exist in RNA molecules. Such heterogeneity has been observed in ionograms of tissue RNA obtained from all major sedimentation zones (9, 12, 13). It is important to note that when whole cytoplasmic RNA is fractionated electrophoretically, the same minor components are present which are noted in electropherograms of fractions after sucrose gradient separation, that patterns are highly reproducible from preparation to preparation, and from tissue to tissue in the rat (9). Though the mean total plasma RNA concentration of 144 mg/liter obtained for the population in this study agreed closely with that reported for a healthy population by Kamm and Smith (7), a considerable range in total concentration was found (50-215 mg/liter) among individuals in the present study. This wide range in total RNA concentration and the variation-in electrophoretic patterns may be due to differences in the metabolic condition of the individual. Broader and more detailed electrophoretie characterizations of plasma RNA are needed to provide additional information that could be beneficial in monitoring the health state of an individual. SUMMARY
Electrophoresis on polyacrylamide-agarose composite gels was used to fractionate plasma RNA from 51 normal individuals. The RNA fractions all migrated as higher molecular weights species in the range of 6-27 S RNA when compared to an Escherichia coli 4 S tRNA reference. In all cases two predominate bands, 3 and 4, were present; with either a single broad diffuse band 1, or two more discrete bands, 1 and 2. Two out of 51 individuals had a fifth more anodic band than band 4. Approximately 76% of the individuals surveyed had an identical banding pattern. Two variants of this pattern comprised 20 and 4% of the total surveyed. Only two females, ages 21 and 23, exhibited the fifth band. No other apparent differences were noted among age and sex groups, nor could any correlation between pattern variants and total plasma RNA concentrations be detected. REFERENCES 1. Javillier, M. and Fabrykant, M., Bull. Sot. Chim. Biol. 13, 1253 (1931). 2. Mandel, P. and Metais, P., C. R. Acad. Sci. 112, 16 (1947). 3. Niemer, H. and Sadtler, E., Klin. Wochenschr. 27, 278 (1949). 4. Fleck, A. and Beg, D., Biochim. Biophys. Acta 42, 333 (1966). 5. Kaznacheev, V. P. and Polyakov, Y. V., Kardiologiga 7, 22 (1967). 6. Kamm, R. C., Dissertation. University of Tennessee, 1970. 7. Kamm, R. C. and Smith, A. G., Clin. Chem. 18, 51%522(1972). 8. Elzen, G. and Kamm, R. C., Camp. Rio&em. Physiol. 4Sa, $81 (1974). 9. Peacock, A. C. and Dingman, C. W., Biochemistry 6, 1818 (1967). 10. McConkey, E. H. and Hopkins, J. W., J. Mol. Biol. 39, 54.5 (1969).
230
GUIN
11. 12. 13. 14.
ET AL.
Granboulan, N. and Scherrer, K., Eur. J. Biochem. 9, 1 (1969). Peacock, A. C. and Dingman, C. W., Biochemistry 7, 668 (1968). Dingman, C. W. and Peacock, A. C., Biochemisrry 7, 659 (1968). Orten, J. M. and Neuhaus, D. W., “Biochemistry.” C. V. Mosby Co.. St. Louis, Missouri, 1970. 15. Lehninger, A. L., “Biochemistry.” Worth Publishers, Inc., New York, New York 197-o.