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The comparative biochemistry of developing Ascaris eggs. III. Flavin adenine dinucleotide extracted from unembryonated eggs
EXPERIMENTAL
PARASITOLOGY
The
16,
Comparative III.
Flavin
I-6 (1964)
Biochemistry Adenine
Dinncleotide
Unemhryonated L. Department
of Physiology, (Submitted
Extracted
Eggs.
from
Eggs1
C. Costello
School of Pharmacy, Baltimore, Maryland for
Ascaris
of Developing
publication,
University
8 February
of Maryland,
1963)
Homogenates of unembryonated Ascuris eggs which were extracted with butanol resulted in the separation of a yellow aqueous phase that exhibited only slight turbidity. Such extracts elicited a fluorometric spectrum similar to flavin. The visible absorption spectrum obtained by spectrophotometric assay of concentrated extracts provided further evidence of the flavin nature of the egg preparations. Aqueous extracts of homogenates of unembryonated eggs stimulated n-amino acid oxidase activity, thereby demonstrating the presence of flavin adenine dinucleotide (FAD). Most of the FAD was protein-bound. It was further established electrophoretically that FAD accounted for essentially all of the detectable flavin. No riboflavin or flavin mononucleotide could be detected by the use of electrophoresis.
Recently, techniques were developed in this laboratory which facilitated a biochemical investigation into the metabolism of Ascaris Zunzbricoides eggs (Costello, 1961; Costello and Brown, 1962). Using homogenizing techniques, the latter authors presented preliminary information concerning the Krebs cycle in unembryonated eggs. As a result of the demonstration of active homogenate preparations, an investigation concerning the terminal electron transport system in Ascaris eggs was initiated. Various extracts of unembryonated eggs were prepared during attempts to identify the components of the transport system. A number of these extracts consistently exhibited a yellow color which appeared to warrant further investigation. The present report is concerned with the identification of the flavin nature of these extracts and the specific detection of flavin adeninedinucleotide (FAD) as the major component. 1 This investigation was supported by National Science Foundation Research Grant G23313.
MATERIALS
AND METHODS
Ascaris lumbricoides var. suum females were obtained from a local abattoir. Decoated eggs were prepared by the homogenizing technique of Costello (1961) with some recent modifications. The harvested eggs were suspended in distilled water and shaken with an equal volume of butanol followed by centrifugation at approximately 2000g for 10 minutes. As a result, the eggs were packed at the bottom of the tube and a lipid pad formed at the butanol-water interface. The packed eggs were resuspended in distilled water and extracted again with butanol. This procedure adequately cleaned the egg preparation of lipid material resulting from homogenization of the uteri. The eggs were then washed several times in distilled water or buffer by repeated centrifugation. Homogenates of eggs were prepared with the aid of a French pressure cell. The eggs suspended in either distilled water or buffer were passed through the cell under a force of 16,000 psi.
2
COSTELLO
The extracts for spectral assay were prepared as follows: The homogenates were centrifuged at 25,000g for 30 minutes. The resulting milky supernatant was filtered through glass wool. An equal volume of butanol was shaken with the supernatant and centrifuged at 25,000g for 10 minutes. The lower aqueous layer exhibited a yellow appearance although somewhat turbid. A lipid pad formed at the butanol-water interface. The aqueous layer was again mixed with butanol and centrifugation repeated. The resulting yellow aqueous extract exhibited only slight turbidity and could be adequately assayed by spectrophotofluorometric methods. Generally, the extracts were prepared in the cold, and centrifugations were conducted in refrigerated centrifuges (International PR-2 and HR-1). When feasible, manipulations were performed in reduced light to minimize photolysis. In some specific experiments modifications of this procedure were utilized. Further purification of the extract was obtained in the following manner: The homogenate obtained from the French pressure cell was deproteinized by placing it in a water bath at 80°C for 15 minutes, followed by high-speed centrifugation and butanol extraction as described above. The extract was then passed through an Amberlite (CG-50) column according to the method of Cerletti and Ipata ( 1960). The eluate was assayed spectrophotofluorometrically. Spectrophotometric assays could not be successfully performed with extracts prepared as described above. Such extracts exhibited too much turbidity in relation to color concentration. Therefore, the flavins were further extracted by the method of Yagi (1962). The extract was prepared by heating and then extracting with butanol as previously described. The author observed that several butanol extractions were required before successful phenol extraction could be accomplished. The flavins were finally concentrated in the aqueous phase of the phenol-ether-
water system and exhibited no turbidity. This extract was assayed spectrophotometritally against an aqueous control and riboflavin standard which were carried through the phenol extraction procedure. Flurometric assays were conducted with a spectrophotofluorometer (Aminco-Bowman) and photometric assays were performed with a recording spectrophotometer (Perkin-Elmer model 350). For enzymatic assay the following procedure was used: The homogenates were prepared in 0.03il4 phosphate buffer (pH 7.2) and heated in a water bath at 80°C for 15 minutes to liberate protein-bound flavins. The homogenates were then centrifuged at 25,000g for 15 minutes. The supernatant fluid was filtered through glass wool and the filtrate assayed manometrically for FAD activity. In studies concerning free and proteinbound flavins, the egg homogenate was divided into two aliquots. One was carried through the entire procedure, whereas the aliquot utilized to ascertain free flavin was not heated prior to centrifugation. Standard Warburg techniques were utilized in the manometric assay of FAD. The assay was according to the method of Huennekens and Felton ( 1957). n-Amino acid oxidase freed of FAD was prepared from hog kidney cortex acetone powder. The activity was measured in air at 37°C. Exact FAD standards were not incorporated since quantitative assays are not reported at this time. For electrophoretic analyses, further extraction and concentration of the flavins were necessary. The filtrate from heated homogenate was shaken with an equal volume of butanol. Following centrifugation (2000g for 10 minutes) the yellow aqueous phase was removed and extracted again with butanol. The procedure was repeated a third time. The aqueous phase was transferred to another tube, saturated with ammonium sulfate, and centrifuged. This step insured relatively complete removal of butanol and provided the
COMPARATIVE
BIOCHEMISTRY
initial step of the subsequent extraction. The flavins were then further concentrated by the phenol extraction method of Yagi ( 1962). During the latter part of this investigation, an additional step was incorporated which resulted in more favorable electrophoretic patterns. The final aqueous phase of the butanol extraction was passed through an Amberlite column and the flavins were eluted with water (Cerletti and Ipata, 1960). The eluate was saturated with ammonium sulfate and treated as described above. This modification apparently removed substances, particularly ultraviolet-absorbing materials, which interfered with the fluorescence of the flavins as well as the migration patterns. The concentrated flavin solution was electrophoresed according to the method of Yagi ( 1962). Electrophoretic separation was performed on cellulose acetate strips with 0.05M phosphate buffer (pH 8.0) by utilizing a current of 2.5 ma per centimeter for 20 minutes. The flavins were detected as fluorescent areas when viewed under an ultraviolet light. Riboflavin, FAD, and flavin mononucleotide (FMN) standards were prepared from commercial preparations (Sigma). RESULTS
Figure 1 presents typical fluorometric spectra of egg extracts compared to a riboflavin standard. The fluorescent maximum for standard and extracts occurred at 520 mu, and all fluorometric assays were conducted at this wavelength. Typically, riboflavin exhibited activation maxima at 270, 370, and 450460 mu. The butanol extracts of eggs exhibited similar peaks at 370 and 460 mu, but the 270 mu peak was absent. Heating the homogenate at 80°C for 15 minutes prior to butanol extraction did not qualitatively alter the fluorescent spectrum. When passed through an Amberlite column, the eluate of extract resulted in a fluorometric spectrum identical to the standard with activation peaks at 270, 370, and 460 mu.
OF AXWis
240
260
EGGS. III
320
360
400
440
460
520
ACTIVATION WAVELENGTH (mb) FIG. 1. A comparison of the fluorescent spectra of egg extracts with riboflavin. The extracts utilized are indicated as follows: Extract = butanol extraction of unheated homogenate; extract-heated = butanol extraction of heated homogenate; eluate of extract = butanol extraction of heated homogenate followed by elution from an Amberlite column. The fluorescent wavelength was 520 mu.
The results of a typical spectrophotometric assay are presented in Fig. 2. The preparation involved a butanol extraction of heated homogenate followed by concentration by the phenol method. The riboflavin standard was carried through the procedure as well. The spectra exhibited identical absorption peaks at 450 mu and troughs at 400 mp. In Table I are presented the results of a typical experiment comparing the effect of --
360
400
440
400
520
Extract Riboflavin
560 600
640
WAVELENGTH (mp) FIG. 2. The absorption spectra of egg extract and riboflavin. Extract was prepared by butanol extraction followed by phenol concentration. Riboflavin was carried through the procedure.
COSTELLO
4
TABLE I A Comparison of the Effect of FAD and Ascaris Egg Extract (Heated) on D-Amino Acid Oxidase Activity Oxygen consumed ui/lS-minute intervals
60
15
15
15
15
Alanine FAD Enzyme
40
42
42
45
169
0
0
0
0
0
6
11
10
13
40
23
23
22
26
94
0
0
0
0
0
0
0
0
0
.__ 0
Enzyme Alanine
Enzyme Alanine Extract
Enzyme Extract
Enzyme Alanine Extract
(c The complete system included 1.4 ml pyrophosphate buffer (0.05 M, pH 8.3), 0.1 ml or.-alanine (1.0 M), 1.0 ml FAD solution (approx. 1 up per milliliter) in the main well; 0.5 ml n-amino acid oxidase solution in the sidearm; 0.2 ml 2070 XaOH in the center well. All systems were prepared in
triplicate, and the values presented are the averages.
FAD and egg extract on n-amino acid oxidase activity. The addition of FAD to the enzyme system markedly stimulated oxygen consumption (300%‘). Egg extract prepared by heating the homogenate at 80°C for 15 minutes also stimulated activity ( 135 p ). The results also demonstrated that activity was linear during the incubation period. Table II presents the composite results of five experiments involving different heated extract preparations. In all cases a marked stimulation of n-amino acid oxidase activity occurred. The variation in respiration between experiments was due to differences in con-
centration of the extracts and enzyme tions. The results
of three experiments
sob-
designed
to compare the effects of heated and unheated on n-amino acid oxidase activity are
extracts
TABLE II oj Ascaris Egg Extracts (Heated) n-An&o Acid Oxidase Activity
OTZ
Oxygen consumed (u1/60 minutes)
Total
System”
FAD
The Eflects
Expt. 1 2 3 4 5
.4lanine Extract Enzyme
Alanine Enzyme
30” 37 89 94 140
4 8 45 40 50
Enzyme 3 0 0 0 0
Q The values presented are the averages of flasks prepared
in triplicate.
presented in Table III. In each experiment, extracts were prepared from equal aliquots of the same egg homogenate and treated identically except for the heat treatment. The results demonstrated in all cases that heated extracts resulted in a greater stimulation than unheated preparations. The results also indicated that the greatest difference between the activity of the two preparations generally occurred during the initial period of the determinations, Figure 3 presents the electrophoretic patterns of extracts compared with riboflavin, F.4D, and FMN. An aliquot of extract was eluted from an Amberlite column and compared with an aliquot in which such treatment was omitted. In both cases a single major peak identical to FAD was observed, although tailing was notably present in the extract not passed through the column. DISCUSSION
Butanol extraction of homogenates of unembryonated Ascaris eggs resulted in the formation of a yellow aqueous phase which could be assayed spectrophotofluorometrically. The fluorescent spectra of such extracts were similar to a riboflavin standard. some preparations did not exhibit acteristic 270 mu activation peak,
Although
the charthis peak
did occur in eluates of extract from Amberlite column. -4pparently the absence of the 270 mlr peak was due to quenching. As should be ex-
COMPARATIVE
A Comparison
BIOCHEMISTRY
OF
Ascaris
TABLE III of the Eflects of Heated and Unheated Extracts Acid Oxidase Activity Oxygen consumed
5
EGGS. III
(WI/lo-minute
on D-Amino Total
intervals)
Expt.
System
10
10
10
10
40
1
Extract (heated) Extract (unheated) Difference
10a
6
6 3
9
31 15
3 (100%)
4
Extract (heated) Extract (unheated) Difference
14 6
Extract (heated) Extract (unheated) Difference
21 9 12 (133%)
2
3
^
5 l(2OoJo)
lt
(400%)
17 10
8
(133%)
5
15 9
7 (70%) 20 13
6 ( 67%) 16 12
7 (55%)
4
80%)
16 (107%)
15 6 9 (lSO%b)
31 30 ( 99%)
(
14 9
(
33%)
5 ( 55%)
61
71 43 28 ( 66%)
a The oxygen consumption has been corrected for endogenous activities, and the values presented represent averages of flasks prepared in triplicate. The values in parentheses represent the per cent increase in activity of the heated over unheated preparations.
FMN
FAD
RF
GE2
3
FIG. 3. A comparison of the e!ectrophoretic patterns of egg extracts and riboflavin, F.4D, and FMN. Pattern 1 represents extract eluted from Amberlite column ; pattern 2 represents extract not passed through column; pattern 3 represents the standards. The outIines presented represent the area of fluoresccnce when viewed with an ultraviolet light.
petted, extracts not passed through the column would contain considerable amounts of ultraviolet-absorbing materials. The author has further observed that the addition of riboflavin to such extracts resulted in the disappearance of the 270 rnp peak from the
riboflavin standard. Spectrophotometric assay further corroborated the flavin nature of the extracts. Concentration of the extract with phenol resulted in a visible spectrum typical of flavins. The fluorometric assays further indicated that flavin derivatives other than riboflavin were the major components of the extract. Cerletti and Ipata (1960) reported that FMN and FAD were easily eluted from an Amberlite column with water, in contrast to the strongly absorbed riboflavin. In the present study this author did not observe any detectable loss of the characteristic yellow color in the eluate from the column, suggesting the absence of considerable amounts of riboflavin. The stimulation of n-amino acid oxidase by heated egg extracts was repeatedly observed and taken as evidence for the presence of FAD. The extracts were deproteinized by heating at 80°C for 15 minutes since the flavins, particularly FAD, do not undergo decomposition under these conditions. The activity observed for extracts prepared in this manner would represent a sum of free and protein-bound FAD. The results of the effects of heated and unheated extracts on n-amino acid oxidase
6
COSTELLO
activity demonstrated that FAD was present in the free form as well as protein-bound (Table III). Initial differences appeared to be the greatest and suggested that some protein-bound FAD was dissociated in the unheated preparations as the experiment progressed. Experimental conditions such as pH and temperature might have resulted in this dissociation. The possibility of some FAD being dissociated from protein by the homogenizing procedure must also be considered. However, the differences observed indicated that protein-bound FAD accounted for more than 50% of the total FAD activity. In addition to the identification by enzymatic assay, electrophoresis provided further evidence of the presence of F.L\D. The electrophoretic pattern obtained from extract passed through an Amberlite column and concentrated by phenol extraction revealed a single spot comparable to FAD. Furthermore, no FMN could be detected, suggesting the presence of, at best, only a relatively slight amount. A comparison of the electrophoretic patterns of extract treated with and without column elution revealed the absence of detectable riboflavin. If riboflavin were present it would have been detected in the extract not passed through the Amberlite column. The observation of the color intensities before and after elution from the column and the resulting electrophoretic patterns would suggest that FAD accounted for nearly all the flavin extracted. An investigation is currently in progress to identify quantitatively the total flavin concentration in eggs as well as relative amounts of derivatives which might be present. The role of FAD in the metabolism of unembryonated Ascaris eggs remains to be established. Costello et al. (1963) reported the absence of physiologically significant cytochrome c oxidase activity in unembryonated eggs. To reconcile the oxygen uptake of these eggs, the authors suggested FAD and flavin oxidase as components of the terminal electron transport system. Such a mechanism has
been described for adult Ascaris (Bueding and Charms, 1952; Bueding et al., 1955). Another important role of the FAD could be related to the significant fat metabolism which occurs in Ascaris eggs (Passey and Fairbairn, 1957). The oxidation of fatty acids involves acyl dehydrogenase, which bears an FAD prosthetic group (Stumpf and Barber, 1960). kxNOWLEDCMENT The author wishes to express his sincere appreciation to the personnel of the Schluderberg-Kurdle Company for their cooperation and assistance in making available the ascarids used in this investigation. REFERENCES BUEDINT., E.. AND CHARMS, B. 1952. Cytochrome c, cytochrome oxidase and succinoxidase activities of helminths. Journal of Biological Chemistry 196, 617-627. BUEDIN~, E.. ENTNER, N., .~ND FARBER, E. 1955. Dissociation of the succinoxidase systems of Ascaris lumbricoides and of rat liver. Biochimica et Biophysics Acta 18, 305-306. CERLETTI, P. AND IPATA, P. 1960. Determination of riboflavine and its coenzymes in tissues. Biochemical Journal 75, 119-124. COSTELLO, L. C. 1961. .4 simplified method of isolating Ascaris eggs. Journal of Parasitology 4. 24. COSTELLO, L. C. AND BROWN, H. 1962. Aerobic metabolism of unembryonated eggs of AscarL lumbricoides. Experimental Parasitology 12, 33-40. COSTELLO, L. C., OYA, H., AND SMITH, W. 1963, The comparative biochemistry of developing Ascaris eggs. I. Substrate oxidation and the cytochrome system in embryonated and unembryonated eggs. Archives of Biochemistry and Biophysics 103, 345-351. HUENNEXENS, F. M., AND FELTOX, S. P., 1957. Preparation and enzymatic assay of FAD and FMN. Methods in Enzymology 3, 950-959. PASSEY, R. F., AND FAIRBAIRX, D. 1957. The conversion of fat to carbohydrate during embryoof nation of Ascaris eggs. Canadian Journal Biochemistry and Physiology 35, jll-j2j. STUMPF, P. K., END BARBER, G. .4. 1960. Comparative mechanisms for fatty acid oxidation. Comparative Biochemistry 1, 75-105, YAGI, K. 1962. Chemical determination of flavins. Methods of Biochemical Analysis 10, 319356.
PARASITOLOGY
The
16,
Comparative III.
Flavin
I-6 (1964)
Biochemistry Adenine
Dinncleotide
Unemhryonated L. Department
of Physiology, (Submitted
Extracted
Eggs.
from
Eggs1
C. Costello
School of Pharmacy, Baltimore, Maryland for
Ascaris
of Developing
publication,
University
8 February
of Maryland,
1963)
Homogenates of unembryonated Ascuris eggs which were extracted with butanol resulted in the separation of a yellow aqueous phase that exhibited only slight turbidity. Such extracts elicited a fluorometric spectrum similar to flavin. The visible absorption spectrum obtained by spectrophotometric assay of concentrated extracts provided further evidence of the flavin nature of the egg preparations. Aqueous extracts of homogenates of unembryonated eggs stimulated n-amino acid oxidase activity, thereby demonstrating the presence of flavin adenine dinucleotide (FAD). Most of the FAD was protein-bound. It was further established electrophoretically that FAD accounted for essentially all of the detectable flavin. No riboflavin or flavin mononucleotide could be detected by the use of electrophoresis.
Recently, techniques were developed in this laboratory which facilitated a biochemical investigation into the metabolism of Ascaris Zunzbricoides eggs (Costello, 1961; Costello and Brown, 1962). Using homogenizing techniques, the latter authors presented preliminary information concerning the Krebs cycle in unembryonated eggs. As a result of the demonstration of active homogenate preparations, an investigation concerning the terminal electron transport system in Ascaris eggs was initiated. Various extracts of unembryonated eggs were prepared during attempts to identify the components of the transport system. A number of these extracts consistently exhibited a yellow color which appeared to warrant further investigation. The present report is concerned with the identification of the flavin nature of these extracts and the specific detection of flavin adeninedinucleotide (FAD) as the major component. 1 This investigation was supported by National Science Foundation Research Grant G23313.
MATERIALS
AND METHODS
Ascaris lumbricoides var. suum females were obtained from a local abattoir. Decoated eggs were prepared by the homogenizing technique of Costello (1961) with some recent modifications. The harvested eggs were suspended in distilled water and shaken with an equal volume of butanol followed by centrifugation at approximately 2000g for 10 minutes. As a result, the eggs were packed at the bottom of the tube and a lipid pad formed at the butanol-water interface. The packed eggs were resuspended in distilled water and extracted again with butanol. This procedure adequately cleaned the egg preparation of lipid material resulting from homogenization of the uteri. The eggs were then washed several times in distilled water or buffer by repeated centrifugation. Homogenates of eggs were prepared with the aid of a French pressure cell. The eggs suspended in either distilled water or buffer were passed through the cell under a force of 16,000 psi.
2
COSTELLO
The extracts for spectral assay were prepared as follows: The homogenates were centrifuged at 25,000g for 30 minutes. The resulting milky supernatant was filtered through glass wool. An equal volume of butanol was shaken with the supernatant and centrifuged at 25,000g for 10 minutes. The lower aqueous layer exhibited a yellow appearance although somewhat turbid. A lipid pad formed at the butanol-water interface. The aqueous layer was again mixed with butanol and centrifugation repeated. The resulting yellow aqueous extract exhibited only slight turbidity and could be adequately assayed by spectrophotofluorometric methods. Generally, the extracts were prepared in the cold, and centrifugations were conducted in refrigerated centrifuges (International PR-2 and HR-1). When feasible, manipulations were performed in reduced light to minimize photolysis. In some specific experiments modifications of this procedure were utilized. Further purification of the extract was obtained in the following manner: The homogenate obtained from the French pressure cell was deproteinized by placing it in a water bath at 80°C for 15 minutes, followed by high-speed centrifugation and butanol extraction as described above. The extract was then passed through an Amberlite (CG-50) column according to the method of Cerletti and Ipata ( 1960). The eluate was assayed spectrophotofluorometrically. Spectrophotometric assays could not be successfully performed with extracts prepared as described above. Such extracts exhibited too much turbidity in relation to color concentration. Therefore, the flavins were further extracted by the method of Yagi (1962). The extract was prepared by heating and then extracting with butanol as previously described. The author observed that several butanol extractions were required before successful phenol extraction could be accomplished. The flavins were finally concentrated in the aqueous phase of the phenol-ether-
water system and exhibited no turbidity. This extract was assayed spectrophotometritally against an aqueous control and riboflavin standard which were carried through the phenol extraction procedure. Flurometric assays were conducted with a spectrophotofluorometer (Aminco-Bowman) and photometric assays were performed with a recording spectrophotometer (Perkin-Elmer model 350). For enzymatic assay the following procedure was used: The homogenates were prepared in 0.03il4 phosphate buffer (pH 7.2) and heated in a water bath at 80°C for 15 minutes to liberate protein-bound flavins. The homogenates were then centrifuged at 25,000g for 15 minutes. The supernatant fluid was filtered through glass wool and the filtrate assayed manometrically for FAD activity. In studies concerning free and proteinbound flavins, the egg homogenate was divided into two aliquots. One was carried through the entire procedure, whereas the aliquot utilized to ascertain free flavin was not heated prior to centrifugation. Standard Warburg techniques were utilized in the manometric assay of FAD. The assay was according to the method of Huennekens and Felton ( 1957). n-Amino acid oxidase freed of FAD was prepared from hog kidney cortex acetone powder. The activity was measured in air at 37°C. Exact FAD standards were not incorporated since quantitative assays are not reported at this time. For electrophoretic analyses, further extraction and concentration of the flavins were necessary. The filtrate from heated homogenate was shaken with an equal volume of butanol. Following centrifugation (2000g for 10 minutes) the yellow aqueous phase was removed and extracted again with butanol. The procedure was repeated a third time. The aqueous phase was transferred to another tube, saturated with ammonium sulfate, and centrifuged. This step insured relatively complete removal of butanol and provided the
COMPARATIVE
BIOCHEMISTRY
initial step of the subsequent extraction. The flavins were then further concentrated by the phenol extraction method of Yagi ( 1962). During the latter part of this investigation, an additional step was incorporated which resulted in more favorable electrophoretic patterns. The final aqueous phase of the butanol extraction was passed through an Amberlite column and the flavins were eluted with water (Cerletti and Ipata, 1960). The eluate was saturated with ammonium sulfate and treated as described above. This modification apparently removed substances, particularly ultraviolet-absorbing materials, which interfered with the fluorescence of the flavins as well as the migration patterns. The concentrated flavin solution was electrophoresed according to the method of Yagi ( 1962). Electrophoretic separation was performed on cellulose acetate strips with 0.05M phosphate buffer (pH 8.0) by utilizing a current of 2.5 ma per centimeter for 20 minutes. The flavins were detected as fluorescent areas when viewed under an ultraviolet light. Riboflavin, FAD, and flavin mononucleotide (FMN) standards were prepared from commercial preparations (Sigma). RESULTS
Figure 1 presents typical fluorometric spectra of egg extracts compared to a riboflavin standard. The fluorescent maximum for standard and extracts occurred at 520 mu, and all fluorometric assays were conducted at this wavelength. Typically, riboflavin exhibited activation maxima at 270, 370, and 450460 mu. The butanol extracts of eggs exhibited similar peaks at 370 and 460 mu, but the 270 mu peak was absent. Heating the homogenate at 80°C for 15 minutes prior to butanol extraction did not qualitatively alter the fluorescent spectrum. When passed through an Amberlite column, the eluate of extract resulted in a fluorometric spectrum identical to the standard with activation peaks at 270, 370, and 460 mu.
OF AXWis
240
260
EGGS. III
320
360
400
440
460
520
ACTIVATION WAVELENGTH (mb) FIG. 1. A comparison of the fluorescent spectra of egg extracts with riboflavin. The extracts utilized are indicated as follows: Extract = butanol extraction of unheated homogenate; extract-heated = butanol extraction of heated homogenate; eluate of extract = butanol extraction of heated homogenate followed by elution from an Amberlite column. The fluorescent wavelength was 520 mu.
The results of a typical spectrophotometric assay are presented in Fig. 2. The preparation involved a butanol extraction of heated homogenate followed by concentration by the phenol method. The riboflavin standard was carried through the procedure as well. The spectra exhibited identical absorption peaks at 450 mu and troughs at 400 mp. In Table I are presented the results of a typical experiment comparing the effect of --
360
400
440
400
520
Extract Riboflavin
560 600
640
WAVELENGTH (mp) FIG. 2. The absorption spectra of egg extract and riboflavin. Extract was prepared by butanol extraction followed by phenol concentration. Riboflavin was carried through the procedure.
COSTELLO
4
TABLE I A Comparison of the Effect of FAD and Ascaris Egg Extract (Heated) on D-Amino Acid Oxidase Activity Oxygen consumed ui/lS-minute intervals
60
15
15
15
15
Alanine FAD Enzyme
40
42
42
45
169
0
0
0
0
0
6
11
10
13
40
23
23
22
26
94
0
0
0
0
0
0
0
0
0
.__ 0
Enzyme Alanine
Enzyme Alanine Extract
Enzyme Extract
Enzyme Alanine Extract
(c The complete system included 1.4 ml pyrophosphate buffer (0.05 M, pH 8.3), 0.1 ml or.-alanine (1.0 M), 1.0 ml FAD solution (approx. 1 up per milliliter) in the main well; 0.5 ml n-amino acid oxidase solution in the sidearm; 0.2 ml 2070 XaOH in the center well. All systems were prepared in
triplicate, and the values presented are the averages.
FAD and egg extract on n-amino acid oxidase activity. The addition of FAD to the enzyme system markedly stimulated oxygen consumption (300%‘). Egg extract prepared by heating the homogenate at 80°C for 15 minutes also stimulated activity ( 135 p ). The results also demonstrated that activity was linear during the incubation period. Table II presents the composite results of five experiments involving different heated extract preparations. In all cases a marked stimulation of n-amino acid oxidase activity occurred. The variation in respiration between experiments was due to differences in con-
centration of the extracts and enzyme tions. The results
of three experiments
sob-
designed
to compare the effects of heated and unheated on n-amino acid oxidase activity are
extracts
TABLE II oj Ascaris Egg Extracts (Heated) n-An&o Acid Oxidase Activity
OTZ
Oxygen consumed (u1/60 minutes)
Total
System”
FAD
The Eflects
Expt. 1 2 3 4 5
.4lanine Extract Enzyme
Alanine Enzyme
30” 37 89 94 140
4 8 45 40 50
Enzyme 3 0 0 0 0
Q The values presented are the averages of flasks prepared
in triplicate.
presented in Table III. In each experiment, extracts were prepared from equal aliquots of the same egg homogenate and treated identically except for the heat treatment. The results demonstrated in all cases that heated extracts resulted in a greater stimulation than unheated preparations. The results also indicated that the greatest difference between the activity of the two preparations generally occurred during the initial period of the determinations, Figure 3 presents the electrophoretic patterns of extracts compared with riboflavin, F.4D, and FMN. An aliquot of extract was eluted from an Amberlite column and compared with an aliquot in which such treatment was omitted. In both cases a single major peak identical to FAD was observed, although tailing was notably present in the extract not passed through the column. DISCUSSION
Butanol extraction of homogenates of unembryonated Ascaris eggs resulted in the formation of a yellow aqueous phase which could be assayed spectrophotofluorometrically. The fluorescent spectra of such extracts were similar to a riboflavin standard. some preparations did not exhibit acteristic 270 mu activation peak,
Although
the charthis peak
did occur in eluates of extract from Amberlite column. -4pparently the absence of the 270 mlr peak was due to quenching. As should be ex-
COMPARATIVE
A Comparison
BIOCHEMISTRY
OF
Ascaris
TABLE III of the Eflects of Heated and Unheated Extracts Acid Oxidase Activity Oxygen consumed
5
EGGS. III
(WI/lo-minute
on D-Amino Total
intervals)
Expt.
System
10
10
10
10
40
1
Extract (heated) Extract (unheated) Difference
10a
6
6 3
9
31 15
3 (100%)
4
Extract (heated) Extract (unheated) Difference
14 6
Extract (heated) Extract (unheated) Difference
21 9 12 (133%)
2
3
^
5 l(2OoJo)
lt
(400%)
17 10
8
(133%)
5
15 9
7 (70%) 20 13
6 ( 67%) 16 12
7 (55%)
4
80%)
16 (107%)
15 6 9 (lSO%b)
31 30 ( 99%)
(
14 9
(
33%)
5 ( 55%)
61
71 43 28 ( 66%)
a The oxygen consumption has been corrected for endogenous activities, and the values presented represent averages of flasks prepared in triplicate. The values in parentheses represent the per cent increase in activity of the heated over unheated preparations.
FMN
FAD
RF
GE2
3
FIG. 3. A comparison of the e!ectrophoretic patterns of egg extracts and riboflavin, F.4D, and FMN. Pattern 1 represents extract eluted from Amberlite column ; pattern 2 represents extract not passed through column; pattern 3 represents the standards. The outIines presented represent the area of fluoresccnce when viewed with an ultraviolet light.
petted, extracts not passed through the column would contain considerable amounts of ultraviolet-absorbing materials. The author has further observed that the addition of riboflavin to such extracts resulted in the disappearance of the 270 rnp peak from the
riboflavin standard. Spectrophotometric assay further corroborated the flavin nature of the extracts. Concentration of the extract with phenol resulted in a visible spectrum typical of flavins. The fluorometric assays further indicated that flavin derivatives other than riboflavin were the major components of the extract. Cerletti and Ipata (1960) reported that FMN and FAD were easily eluted from an Amberlite column with water, in contrast to the strongly absorbed riboflavin. In the present study this author did not observe any detectable loss of the characteristic yellow color in the eluate from the column, suggesting the absence of considerable amounts of riboflavin. The stimulation of n-amino acid oxidase by heated egg extracts was repeatedly observed and taken as evidence for the presence of FAD. The extracts were deproteinized by heating at 80°C for 15 minutes since the flavins, particularly FAD, do not undergo decomposition under these conditions. The activity observed for extracts prepared in this manner would represent a sum of free and protein-bound FAD. The results of the effects of heated and unheated extracts on n-amino acid oxidase
6
COSTELLO
activity demonstrated that FAD was present in the free form as well as protein-bound (Table III). Initial differences appeared to be the greatest and suggested that some protein-bound FAD was dissociated in the unheated preparations as the experiment progressed. Experimental conditions such as pH and temperature might have resulted in this dissociation. The possibility of some FAD being dissociated from protein by the homogenizing procedure must also be considered. However, the differences observed indicated that protein-bound FAD accounted for more than 50% of the total FAD activity. In addition to the identification by enzymatic assay, electrophoresis provided further evidence of the presence of F.L\D. The electrophoretic pattern obtained from extract passed through an Amberlite column and concentrated by phenol extraction revealed a single spot comparable to FAD. Furthermore, no FMN could be detected, suggesting the presence of, at best, only a relatively slight amount. A comparison of the electrophoretic patterns of extract treated with and without column elution revealed the absence of detectable riboflavin. If riboflavin were present it would have been detected in the extract not passed through the Amberlite column. The observation of the color intensities before and after elution from the column and the resulting electrophoretic patterns would suggest that FAD accounted for nearly all the flavin extracted. An investigation is currently in progress to identify quantitatively the total flavin concentration in eggs as well as relative amounts of derivatives which might be present. The role of FAD in the metabolism of unembryonated Ascaris eggs remains to be established. Costello et al. (1963) reported the absence of physiologically significant cytochrome c oxidase activity in unembryonated eggs. To reconcile the oxygen uptake of these eggs, the authors suggested FAD and flavin oxidase as components of the terminal electron transport system. Such a mechanism has
been described for adult Ascaris (Bueding and Charms, 1952; Bueding et al., 1955). Another important role of the FAD could be related to the significant fat metabolism which occurs in Ascaris eggs (Passey and Fairbairn, 1957). The oxidation of fatty acids involves acyl dehydrogenase, which bears an FAD prosthetic group (Stumpf and Barber, 1960). kxNOWLEDCMENT The author wishes to express his sincere appreciation to the personnel of the Schluderberg-Kurdle Company for their cooperation and assistance in making available the ascarids used in this investigation. REFERENCES BUEDINT., E.. AND CHARMS, B. 1952. Cytochrome c, cytochrome oxidase and succinoxidase activities of helminths. Journal of Biological Chemistry 196, 617-627. BUEDIN~, E.. ENTNER, N., .~ND FARBER, E. 1955. Dissociation of the succinoxidase systems of Ascaris lumbricoides and of rat liver. Biochimica et Biophysics Acta 18, 305-306. CERLETTI, P. AND IPATA, P. 1960. Determination of riboflavine and its coenzymes in tissues. Biochemical Journal 75, 119-124. COSTELLO, L. C. 1961. .4 simplified method of isolating Ascaris eggs. Journal of Parasitology 4. 24. COSTELLO, L. C. AND BROWN, H. 1962. Aerobic metabolism of unembryonated eggs of AscarL lumbricoides. Experimental Parasitology 12, 33-40. COSTELLO, L. C., OYA, H., AND SMITH, W. 1963, The comparative biochemistry of developing Ascaris eggs. I. Substrate oxidation and the cytochrome system in embryonated and unembryonated eggs. Archives of Biochemistry and Biophysics 103, 345-351. HUENNEXENS, F. M., AND FELTOX, S. P., 1957. Preparation and enzymatic assay of FAD and FMN. Methods in Enzymology 3, 950-959. PASSEY, R. F., AND FAIRBAIRX, D. 1957. The conversion of fat to carbohydrate during embryoof nation of Ascaris eggs. Canadian Journal Biochemistry and Physiology 35, jll-j2j. STUMPF, P. K., END BARBER, G. .4. 1960. Comparative mechanisms for fatty acid oxidation. Comparative Biochemistry 1, 75-105, YAGI, K. 1962. Chemical determination of flavins. Methods of Biochemical Analysis 10, 319356.