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Amino acid analyses of the proteins of two strains of potato virus X
VIROLOGY
18, 79-88 (1962)
Amino
Acid
Analyses
J. G. SHAW,l Research
Branch,
of the
Proteins Virus X
of Two
M. E. REICHMANN, Research
AND
May
of
Potato
D. L. HATT
Station, Canada Department Vancouver, B. C.?
Accepted
Strains
of Agriculture,
1, 1969
The amino acid compositions of the proteins isolated from two biologically similar strains of potato virus X have been determined by chromatography on columns of Amberlite IR-120. Independent estimations were made for tryptophan and cysteine, which was determined after oxidation to cysteic acid. The extent of variation of each amino acid was estimated by determining a confidence limit (P = 0.05) from the standard deviation of the mean. With both proteins, losses of threonine and serine and increases in valine, leucine, and isoleucine were observed as hydrolysis proceeded. The analyses indicate that both virus proteins (subunit molecular weight about 52,000) are composed of 463 amino acid residues of which about 40% consist of alanine, threonine, and aspartic acid (asparagine). The two virus proteins had remarkably similar amino acid compositions, the only statistically significant differences involving one aspartic acid (asparagine) and one methionine residue. INTRODUCTION
fate and resuspension in water followed by dialysis against water. This procedure favors aggregation of PVX particles and makes very difficult the removal of small amounts of nonvirus material (Bawden and Kleczkowski, 1948; Kleczkowski and Nixon, 1950). It seemed appropriate, therefore, to study the amino acids of this virus by analyzing hydrolyzates of the protein isolated by dissociation of highly purified, nonaggregated virus. A method for preparing nonaggregated PVX in a highly purified condition has been described in a previous report from this laboratory (Reichmann, 1959). More recently, the protein moiety of the virus has been isolated and found to have a subunit molecular weight of 52,000 * 2000 (Reichmann, 1960). An investigation of the amino acid contents of t,he proteins of two strains of PVX prepared by the same procedure forms the subject of this communication.
The amino acid content of potato virus X (PVS) has been the subject of two reports in recent years. Baudart (1957) investigated two European st’rains of this virus by paper chromatography and electrophoresis. More recently, Shaw and Larson (1962) presented the results of a preliminary study of the amino acid composition of another strain of PVX analyzed by chromatography on 8% cross-linked Amberlite IR-120 resin. In both cases, analyses were performed on hydrolyzates of preparations of the intact virus. Acid hydrolysis of nucleoprotein rather than simple protein results in the destruction of some amino acids and contributes to the formation of amino acid-like compounds during nucleic acid degradation (Markham and Smith, 1949). In addition, the analyses of Baudart (1957) were made on hydrolyzates of virus preparations purified by repeated precipitation with ammonium sul-
MATERIALS
1 Present address : Plant Research Institute, Central Experimental Farm, Ottawa, Ontario. ‘Contribution No. 40.
AND
METHODS
Viruses. Two strains of PVX, both apparently of the ringspot type, were prepared 79
80
SHAW, REICHMANN,
in quantities sufficient for amino acid analyses. One of them, strain C (Wright and Hardy, 1961), was isolated from potato in British Columbia, it is the same as that used in previous studies at this laboratory (Reichmann, 1959). It produces small, white, irregular rings on the inoculated leaves of Nicotiana tabacum L. (variety Haronova) , and rapidly becomes systemic, resulting in the formation of a severe mot,tle on the uninoculated leaves. The ot,her strain was the ringspot strain originally isolated from potato in Wisconsin (Ladeberg et al., 1950). It incites small, white, more regular rings on the inoculated leaves of tobacco and slowly becomes systemic, at which time a mild, irregular mottle appears on the upper leaves. To avoid compounding the already confusing situation in the nomenclature of PVX strains, the original designations-the C strain and the ringspot, strain -will be used throughout this report. Virus material was isolated from systemically infected tobacco plants grown in a room at 18°C with 120-160 foot-candles of fluorescent light for 16 hours per day. The symptoms mentioned above are those incited by each strain under these carefully controlled conditions. Virus samples were prepared by the method of Reichmann (1959)) which has consistently yielded highly purified, nonaggregated material. Amino acid analyses of virus proteins. Amino acid analyses were performed on hydrolyzates of t,he proteins isolated from both strains of the virus. Four different preparations of the C strain and three of the ringspot st.rain were used in the complete determinations. The protein was separated from the nucleic acid by treatment of the virus (in 0.005 M sodium citrate) with 2 M guanidine hydrochloride (Reichmann, 1960). After removal of the nucleic acid precipitate by centrifugation, the reagent was removed by dialysis against distilled water (brought to pH 7.5 with NaOH) at 2” for about 10 hours. Exhaustive dialysis was avoided since it usually led to severe aggregation of the protein. The dialyzed solution was centrifuged for 90 minutes in a refrigerated Spinco model L ultracentrifuge at 40,000 rpm; the small pellet, presumably incompletely split virus, was discarded. The
AND HATT
weight of protein in solution was determined by drying aliquots in a vacuum desiccator over PaOa and then at 107” to constant weight. Control solutions of guanidine hydrochloride in 0.005 M sodium citrate were dialyzed, centrifuged and weighed in the same manner. The small amounts of solute remaining in these solutions were subtracted from the weights of the appropriate protein samples. Presumably some ions interacted with and remained bound to the protein, but the error from this source was likely quite small. For hydrolysis, weighed aliquots of protein solution (about 10 mg) in Pyrex glass tubes were made 6 N in HCI by adding concentrated acid. The final volume was approximately 0.3 ml 6 il: HCl per milligram of protein. The tubes were sealed under vacuum and the samples were hydrolyzed for periods of 12, 24, and 48 hours in an oven at 107”. The use of different times of hydrolysis was deemed necessary because of evidence that amino acids differ considerably in their degree of stability and rates of release during hydrolysis (Hill et al., 1959). The hydrolyzates were light yellow in color; little or no insoluble humin was noticed. Excess HCl was removed by several cycles of evaporation in vacua at 40” and dilution in water, and the samples were finally made to volume in 0.2 M cit,rat.e buffer, pH 2.2 (Moore et al., 1958). Aliquots of the hydrolyzates equivalent to 1.5-2.0 mg protein were chromatographed on 15- and 150-cm columns of Amberlite IR-120 resin as described by Moore et al. (1958). Effluent fractions of 2.0 ml were collected with a volumetric fraction collector (Gilson Medical Electronics), a check on the weight of fractions being made at various intervals with tared tubes. The amino acid content of each fraction was determined by the photometric ninhydrin method of Moore and St.ein (1954) with the exception that reduced ninhydrin was formed by using stannous chloride (Moore and Stein, 1948) instead of by adding hydrindantin directly to the reagent mixture. The yield of each amino acid was determined by simple addit’ion of the color yields within each peak after subtraction of the appropriate baseline color. Carefully pre-
AMINO
ACID
ANALYSES
RESULTS
ASD
DISCUSSIOX
The analytical data from 12-, 24- and 48hour hydrolyzates of the two strain protcins are presented in Tables 1 and 2. Each analysis represents a single determination on the preparation indicated. The values obtained for all three periods of hydrolysis
TBBLE
1
(C STR.UN) AFTER DIFFERENT
-
81
PROTEISS
removal of excess performic acid (Moore, 1961). The excess SO, was removed with gentle swirling of the solution under partial vacuum to avoid severe foaming during subsequent evaporation to dryness. Aliquo ts of the hydrolyzate were chromatographed on 150-cm columns. The cysteic acid content was estimated by reference t’o the molar quantities of stable amino acids (aspartic acid, glutamic acid, and alanine) determined in the same analysis and for which the contents were known from analyses of unoxidized samples of the protein. The result,s were corrected for the 10% loss in recovery of cysteic acid.
pared solutions of known concentration of each amino acid (California Corporation for Biochemical Research) and the appropriate blanks were analyzed along with the effluent fractions in order to convert optical density readings to micromoles of amino acid. The recoveries of amino acids from freshly prepared columns were determined with the use of Spinco Calibration Mixture No. 1 (Beckman Instruments, Inc., Spinco Division) ; similar checks were made periodically during the course of the subsequent analysts. The tryptophan contents of the virus proteins were determined by ,V-bromosuccinimide cleavage of tryptophyl peptide bonds (Pat~chornik et al., 1958). Combined cyst& and cystcine determinations were made on separate samples of the proteins oxidized with performic arid prior to hydrolysis (Schram et aZ., 1954). At t#he completion of t’he oxidation reaction, sodium bisulfite was added to facilitate the
AMINO ACID REVOVERIES FROMPROTEIN OF PVX
OF PVX
TIMES OF HYDROLYSIS
Grams per 100 g protein Amino acid
I-
24 Hour
12 Hour
Average values f SD
48 Hour
I-
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine” Isoleucine Leucine Tyrosine Phenylalanme Lysine Histidine hrginine Ammonia
1
2
1
2
3
11.05 12.69 6.15 9.57 7.80 3.43 12.89 5.29 4.33 5.02 4.64 1.54 7.38
10.97 13.30 6.21 9.13
10.81 12.47 5.79 9.61 7.72 3.34 12.85 5.96 4.46 5.57 4.84
10.42 12.60 5.71 9.38 7.40 3.26 13.66 5.98 4.37 5.35 4.62 1.45 6.95 6.28 1.29 6.14 1.56
10.74 12.60 5.86 9.48 7.47 3.41 12.96 5.77 4.55 5.43 4.97 1.53 7.65 6.11 1.17 6.15 1.49
-
3.40 13.67 5.55 4.53 4.86 4.58 1.52 7.22
7.39 6.39 1.25 6.21 1.51
u Values obtained by extrapolation to zero time (see Fig. 1). h Yields corrected for 5!% loss (Moore et al., 1958). c 48.hour value used as average. d 24 and 48-hour values used in average.
1
2
6.38 I .21 6.23 1.80
10.87 11.56 5.30 9.48 7.i8 3.3-l 13.52 6.14 4.56 5.55 4.98 1.56 6.99 6.30 1.26 6.50 1.74
3
-
6.41 1.19 6.16 1.69
10.81 13.5oa 6.43a 9.44 i.63 3.36 13.26 6.14r 4.47 5.48 4.9@ 1.52 7.26 6.31 1.23 6.23 1.3w
zt 0.09
rt & f zk
0.07 0.08 0.03 0.16
f f
0.04 0.05d
III + zt f f
0.02 0.11 0.05 0.02 0.06
82
SHAW,
REICHMANN,
AND
TABLE
HATT
2
AMINO ACID RECOVERIES FROM PROTEIN OF PVX (RINGSPOT STRAIN) AFTER DIFFERENT TIMES OF HYDROLYSIS
-
-
Grams per 100 g protein
/ Amino acid
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine” Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Ammonia
12 Hour
11.23 13.29 6.26 9.40 7.34 3.41 14.03 4.66 3.97 5.04 4.71 1.37 7.00
24 Hour
11.31 13.20 6.21 9.25
11.00 12.57 5.80 9.66 7.38 3.49 13.22 5.85 4.10 5.48 4.82 1.49 7.34 6.27 1.14 6.33 1.50
7.22 3.63
13.79
-
-
5.15 4.12 4.82 4.56 1.41 7.23
-
11.28 12.56 6.12 9.19 7.15 3.28
13.09 5.28 4.23 5.61 4.60 1.38 7.33 6.35 1.21 6.35 1.58
48 Hour 2
2
1
11.20 12.58 6.04 9.44 7.21 3.18 13.24 5.94 4.12 5.47 4.49 1.53 7.34 6.33 1.21 6.41 1.44
11.78
11.07 11.45
12.93 6.04 9.25 6.89 3.34 13.16 5.90 4.37 5.59 5.20 6.39 1.21 6.25 1.55
11.43
5.56 9.16 7.15 3.58 13.51 6.21 4.20 5.38 5.13 1.40 7.55 6.47 1.21 6.10 1.84
-
1
--
5.56 9.43 6.87 3.49 13.45 6.15 4.21 5.41 5.05 1.40 6.98
-
-
4verage values f
SD
2 11.40 11.77 5.67 9.33 7.38 3.34
11.28 13.80a 6.43a 9.35 7.18 3.42 13.38 6.15 4.17 5.47 5.06 1.43 7.25 6.35 1.20 6.26 1.29=
12.89 6.09 5.38 5.01
6.29 1.23 6.10
1.64
zt 0.08
+ f f + + z!x rt f f f rt
0.05 0.06 0.05 0.12 0.03” 0.04 0.04d 0.04~ 0.02 0.08 0.03
f
0.01
f
0.05
-
a Values obtained by extrapolation to zero time (see Fig. 1). b Yields corrected for 5yo loss (Moore et al., 1958). c 48-hour values used in average. d 24. and 48.hour values used in average.
were included in the average when no significant increase or decrease with time was detected. The standard deviation of the mean (SZ) is included for each amino acid except threonine and serine (and, in the C strain, valine and leucine, for each of which only one value was used). With both proteins, the threonine and serine recovered decreased noticeably as time of hydrolysis increased. The concentrations of these amino acids were calculated by extrapolation of the values to zero time by the method of least squares, assuming a linear decrease of each amino acid with time (Fig. 1). After 24 hours’ hydrolysis, serine and threonine had decreased by 9.0% and 7.1%, respectively, in the C strain protein, and by 6.4% and 8.2%, respectively, in the ringspot strain protein. In neither case was
close. These different rates suggest the presence of adjacent residues in the proteins with differing degrees of susceptibility t,o hydrolytic attack. The values for methionine were corrected for the 5% loss reported by Moore et al. (1958). None of the other amino acids appeared to suffer destructive losses with prolonged hydrolysis. In both proteins, valine, leucine, and isoleucine yielded greater recoveries as the hydrolysis time increased. Isoleucine appeared to have been completely released after 24 hours and the values for the 24- and 48-hour hydrolyzates were used in the average. With valine and isoleucine, increases between 24 and 48 hours were obtained and only the 48-hour values were averaged. The increases in amounts of these three
the rate
drolysis are in accord with many previous observations on the relatively greater stability of the peptide bonds associated with
of decomposition
similar
between
strains though, especially in the case of serine, the extrapolated values were very
amino
acids
obtained
after
prolonged
hy-
AMINO
2 Sz 8cl4= t7
ACID ANALYSES
:.! SERINE
32-
.
1 0’
83
OF PVX PROTEISS
AMMONIA I
I
I
12
24
36
+-----+ IAMMONIA
I
I
I
48 0 12 TIME OF HYDROLYSIS (HOURS)
I
I
24
36
I 48
FIG. 1. Recovery of threonine, serine, and ammonia from acid hydrolyzates of potato virus X protein as a function of duration of hydrolysis. (A) C strain protein. (B) Ringspot strain protein. Lines represent the best fit to the data according to the method of least squares.
these amino acids (Hill et al., 1959). The recoveries of arginine appeared to increase as hydrolysis proceeded with the C strain and to decrease with the ringspot strain. These changes were slight, however, and all the values were included in the averages. Several analyses of unoxidized protein samples showed small, inconsistent peaks in the region of cystine, but, the highest of these amounted only to about 0.870 halfcystine. On t’he other hand, in samples oxidized with performic acid prior to hydrolysis the cysteic acid peak was well defined and separated from the other amino acid peaks. The molar yields of cysteic acid were determined by comparison with t,hose for aspartic acid, glutamic acid, and alanine in the same analyses. A mean value of 1.26 t 0.03 isi: = 0.03) g cysteine per 100 g protein was obtained for the C strain and agrees reasonably well with the result of Reichmann and Hatt (unpublished data) determined by the same procedure. Similar analyses on the ringspot strain yielded an average value of 1.12 i- 0.03 (sf = 0.02) g cysteine per 100 g protein. The lnain diffi-
culty in assessing the significance of these data arises from the necessity of analyzing large amounts of hydrolyzed protein in order to obtain cysteic acid peaks large enough to be properly evaluated. Under these circumstances, the color yields of the reference amino acids are very high and the baselines occasionally less steady than usual. This method of determining the cysteic acid content, however, proved to be more reproducible than that used for the other amino acids in unoxidized protein samples. In a previous study on the C strain of PVX, Reichmann and Hatt (1961) found 8 sulfhydryl groups per prot’ein subunit by means of p-chloromercuribenzoate substitution. Difficulties were encountered in the determinations because of the formation of emulsions with the dithizone-chloroform reagent. There is also the possibility that some nonspecific absorption of p-chloromercuribenzoate by the precipitated and denatured protein occurred. These factors may account for the discrepancy in cysteine determinations bet,ween this value and the data reported in the present investigation. On the
84
SHAW, REICHMANN,
other hand, the possibility of a more extensive destruction of cysteine during performic acid oxidation should be mentioned. In spite of this discrepancy, however, these investigations suggest that cysteine was the sole source of cysteic acid and that the virus protein contains no cystine. In terms of protein structure this would indicate that the subunit is a single polypeptide chain. Since tryptophan undergoes almost complete destruction during acid hydrolysis, the method of Patchornik et al. (1958)) involving the titration of C-tryptophyl peptide bonds cleaved by N-bromosuccinimide, was used. Average values of 3.45 (se = 0.07) and 3.34 (sz = 0.02) g/100 g virus protein were obtained for the C strain and ringspot strain, respectively, from analyses of three different preparations of each strain. The amide ammonia contents of the virus proteins were estimated by extrapolation of the values from 24- and 4%hour hydrolyzates to zero time by the method of least squares (Fig. 1). Values of 1.29% for the C strain and 1.30% for the ringspot strain were obtained. These values may exceed the actual amounts of ammonia liberated from amide groups during hydrolysis since Chibnall et al. (1958) found that various native proteins contain small amounts of free ammonia. Analyses of the virus proteins for free ammonia were not made, and the above estimates for amide ammonia must be regarded as maximum values only. In each case, the excess ammonia after 48 hours’ hydrolysis (calculated from the least squares values) was 0.026 mole/100 g protein. The amounts of threonine and serine destroyed during 48 hours hydrolysis were equivalent, to 0.027 mole for both proteins. As with other proteins (Rees, 1946; Smith and Stockell, 1954; Smith et al., 1954) it appears that the amount of ammonia liberated in excess of that at zero time of hydrolysis was due to the destruction of threonine and serine. The breakdown of cysteine and tryptophan apparently did not contribute to this excess ammonia. As reported by Moore et al. (1958)) analyses of standard calibration mixtures yielded recoveries of 100 f 3% for all the amino acids except methionine for which the recovery was 95%. Before a freshly poured
AND HATT
150-cm column would yield complete recoveries, however, it was necessary to pass alternating quantities of NaOH and citrate buffer (pH 3.25) through it six to eight times as in the regeneration cycle (Moore et al., 1958). The separation of peaks on the chromatograms was excellent except for threonineserine and tyrosine-phenylalanine. For the quantitative estimation of threonine and serine the color yield in the tube at the lowest point between the two peaks was divided equally between the two amino acids or in proportion to the yields in hhe tubes immediately preceding and following it. Since this amount constituted only a small percentage of the total color yields of each of the two peaks, the error involved, was probably slight. The tyrosine and phenylalanine determinations were made in the same manner. In the case of tyrosine, however, the error may be more appreciable because of its low content in the hydrolyzates. The total recoveries of amino acid residues from each strain protein were 96-97%, excluding the ammonia (Tables 3 and 4). Several factors may be responsible for this somewhat low recovery in weight. Small errors in the integration of the areas under the peaks of some amino acids are probably unavoidable as a result of variat,ions in the baseline. In general, the chromatoconsistently stable grams demonstrated baselines except in the valine-methionine region where a change in the pH of the eluent occurs. Because of the limited amounts of purified PVX that could be extracted from any group of plants, no more than about 10 mg of protein could be used in any single hydrolysis tube. With larger quantities of protein it is likely that the small losses inherent in the manipulations between hydrolysis and application of the samples to the columns would have been less significant. Some losses may also have been introduced during the manual procedure of ninhydrin color development. It is quite probable that the automatic recording method of amino acid analysis (Spackman et al., 1958) introduces less error in the determinations than does the manual method used in t’his study. Un-
.4MINO
ACID ANALYSES TABLE
Amino acid
COMPOSITION AND MOLECULAR
g Amino acid/lOOg protein”
g Amino cid residue/ OOgprotei; 1
10.81 13.50
9.35 11.46
6.43 9.44 7.63 3.36 13.26 6.14 4.47 5.48 4.98 1.52 7.26 6.31 1.23 6.23 3.45 1.26
5.33 8.28 6.44 2.55 10.58 5.20 3.93 4.73 4.30 1.37 6.47 5.53
-1.. Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alaninc Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Hist,idine Arginine Tryptophan” Cysteine c Ammonia
3
WEIGHT OF PVX
-
-
I
85
OF PVS PROTEINS
Minimal mol. wt.
(C STRAIN) -
Calculated no. Assumed residues (mol. I10. residues lalculated mol. wt. wt. = 51,140)’ -
1.30
1.09 5.59 3.15 1.07 1.22d
1,231
41.55 f 57.99 31.30
882 1,634
1,559 1,508 2,237 672
1,906 3,338 2,392 2,632 11,910 2,275 2,318 12,582 2,794 5,911 9,615 1,314
32.81 33.92 22.86 76.11 26.83 15.32 21.38 19.43 4.29 22.48 22.06 4.06 18.30 8.65 5.32 38.92
0.90
h 0.62 + 1.03 f 0.44 f 2.36 f f
0.35 0.65
f f f I!C zk f r!z
0.15 0.87 0.41 0.16 0.42 1.21 0.74
96.42
Totals
51,702 51,156
42 58 31 33 34 23
~
76
I
27 15 21 19 4 22 22 4 18 9 5 39d 463
50,654 51,447 51,272 51,451 51,072 51,462 50,070 50,232 50,008 47,640 50,050 50,996 50,328 50,292 53,199 48,075 51,246
/
(Av.
= 50,654)
a From Table 1. b Values of 3.46% (preparation 2), 3.577c (preparation 3), and 3.33@j, (preparation 4) obtained by method of Patchornik et al. (1958). Average = 3.45 (sz = 0.07) g tryptophan per 100 g protein. c Determined as cysteic acid in performic acid oxidized protein (Schram et al., 1954; Moore, 1961). Values ohtained by comparison to reference amino acids (see text) averaged to determine content,. d Not included in totals. e With tsi: at P = 0.05, n-l degrees of freedom. fortunately, only a single analysis of the C strain after 48 hours’ hydrolysis was obt,ained, thus the values for those amino acids increasing or decreasing as hydrolysis proceeded may be somewhat inaccurate for this strain. In addition, a period of hydrolysis longer than 48 hours might have yjclded further recoveries of valine and leucure and thus contributed to a more com-
plete analysis of each strain protein. The molecular weight of the protein subunit of the C strain of PVX has been established as 52,000 * 2000 from sedimentation-
diffusion
measurements
in this laboratory
(Reichmann, 1960). The discrepancies between this value and that of 74,000 calculated from C-terminal end-group analysis
by hydrazinolysis already
(Niu et cd., 1958) have
been discussed
(Reichmann,
1960).
Analysis of a sample of the ringspot strain protein in 2 M guanidine hydrochloride demonstrated a sedimentation rate in the Spinco model E analytical ultracentrifuge similar to that of the C strain. This suggested a subunit of similar or equal size. From the amino acid analyses, it was found that values slightly
lower than 52,000 better
fitted the data when considered as assumed molecular weights of the proteins. Thus, the values 51,140 and 51,200 for the C strain and ringspot
strain,
respectively,
were used
for calculating the number of residues prcsent in each subunit (Tables 3 and 4). On this basis, the general averages of 50,654 (C strain) and 50,999 (ringspot strain), which were obtained from t’he molecular weights calculated from all the amino acids,
86
SHAW,
REICHMANN,
AND
TABLE COMPOSITION
Amino acid
Aspsrtic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan” Cyst,eine c Ammonia Totals
AND MOLECULAR -
4
WEIGHT
g Amino acid/100 g proteina
g Amino cid residue I 00 g protein n
Minimal mol. wt.
11.28 13.80 6.43 9.35 7.18 3.42 13.38 6.15 4.17 5.47 5.06 1.43 7.25 6.35 1.20 6.26 3.34 1.12 1.29
9.75 11.71 5.33 8.21 6.06 2.60 10.67 5.20 3.67 4.72 4.37 1.29 6.46 5.57 1.06 5.61 3.05 0.95 1.21d
1,180 863 1,634 1,573 1,603 2,194 666 1,906 3,575 2,397 2,589 12,649 2,278 2,301 12,938 2,784 6,105 10,817 1,325
96.28
I
HATT
OF PVX
-
(RINGSPOT
Calculated no. residues (mol. wt. = 51,203)e 43.39 59.33 31.34 32.55 31.94 23.34 76.88 26.86 14.32 21.36 19.78 4.05 22.48 22.25 3.96 18.39 8.39 4.73 38.64
I
f
STRAIN)
Assumed I10. residues
0.76
lalculated
43 59 31 33 32 23 77 27 14 21 20 4 22 22 4 18 8 5 39d
rk 0.42 f 0.65 z!z 0.77 f 1.59 f 1.05 f 0.34 & 0.34 & 0.96 & 0.17 f 0.58 f 0.24 f 0.11 f 0.41 f 0.41 It 0.20
463
mol. wt.
50,740 50,917 50,654 51,909 51,296 50,462 51,282 51,462 50,050 50,337 51,780 50,596 50,116 50,622 51,752 50,112 48,840 54,085 51,675 (Av.
= 50,999)
-
a From Table 2. b Values of 3.30% (preparation I), 3.33% (preparation 2), and 3.38c/, (preparation 3) obtained by method of Patchornik et al. (1958). Average = 3.34 (sf = 0.02) g tryptophan per 100 g protein. c Determined as cysteic acid in performic acid oxidized protein (Schram et al., 1954; Moore, 1961). Values obtained bv comDarison to reference amino acids (see text) averaged to determine content. d Not included in totals. e With tsi: at I’ = 0.05, n-l degrees of freedom
are in close agreement with the molecular weight from physical measurements. The estimations of the number of residues of each amino acid in the two proteins are presented in Tables 3 and 4. From the % values (Tables 1 and 2) and n - 1 degrees of freedom, 95% confidence limits (ts, ) have been calculated. In the C strain protein, only one possible integer is within the calculated limits of error for glutamic acid, glycine, methionine, lysine, histidine, and arginine. Similarly, for proline, met,hionine, tyrosine, histidine, arginine, and tryptophan in the ringspot strain, only one possible integer is included within the 95% confidence intervals. An integral number of residues lying within the limits of error was not found for five amino acids (tyrosine in the C strain, and glutamic acid, isoleucine, lysine, and half-cystine in the ringspot
strain). Most of these, however, lie considerably closer to one integer than the other, and the nearest whole number has been assigned as the assumed number of residues per subunit. For the remaining amino acids in each strain, most of which are present in excess of 20 residues, there is more than one possible integer. High tsi values were obtained for several amino acids and consequently increased the number of possible integers. Proline gives a color with low extinction with ninhydrin reagent and analysis of this amino acid is somewhat less precise. Fewer data were available for the valine, isoleucine, and leucine calculations since the recoveries of these amino acids increased as hydrolysis proceeded. The independent determinations of cysteine and tryptophan were also based on fewer results than those of amino acids calcula,ted
AMINO
ACID
rlNALlrSES
after several hydrolysis times. Accurate intcgrat,ion of the aspartic acid and alanine peaks was more difficult because of the very large amounts of these amino acids present in the prot,eins. The analyses indicate that the PVX protein subunit is composed of 463 amino acid residues or slightly more when the 3-4s loss of recovery is considered. None of t,he common amino acids appear to be absent from either of the strain proteins examined nor do any occur to the extent of only one rcsiduc per subunit. Each strain contains less than 10 residues each of cysteine, tyrosine, tryptophan, and histidine per protein subunit. Of interest is the high content of alaninc, which comprises about 16.5% of the number of residues in each strain. Almost 40:/c of t,he number of residues in each protein is made up of the three amino acids alanine, threonine, and aspartic acid (asparagine). Based on the extrapolated ammonia values, each strain contains a maximum of 39 amide residues though, as mentioned earlier, some of these groups may represent free ammonia in the proteins. Chibnall et al. (1958) found errors in reported values for amide nitrogen in various proteins amounting to as much as 10% as a result. of the presence of free ammonia. With the possibility of this source of error in mind, it would appear that about half the aspartic and glutamic acid residues obtained after hydrolysis of the PVX strain proteins were originally in the amide form. These, however, cannot be assigned as definite numbers of asparaginyl or glutaminyl residues. The data of Shaw and Larson (1962) on another strain of PVX demonstrate a proportionately similar pattern in amino acid composition to those reported here though the percentage recoveries were somewhat less. It would be difficult, however, to compare the results of the two studies since the former involved a considerably less complete investigation of amino acid content. The results of the study by Baudart (1957) appear to be less similar to those reported herein but the different m&hods of preparation and analysis and the lower recoveries in the former make any comparisons unfeasible. In neither of the earlier reports were independent analyses made for cry-
OF PVX
PROTEINS
87
teine content, nor were different times of hydrolysis used to account fully for the labile and slowly released amino acids present. The analyses were made on hydrolyzates of intact virus and the higher glycinc yields obtained by Shaw and Larson (1962) can likely be attributed to the degradation of purines. It would seem, therefore, that the present analyses offer the most. complete data on the amino acid compositions of the PVX strains thus far examined. A comparison of the amino acid compositions of the C strain and ringspot strain proteins demonstrates a remarkable similarity between the two. Only in the cases of aspart’ic acid and methionine were differences significant at the 5% confidence level obtained. From the statistical analysis, the C strain would appear to contain one fewer aspartic acid (asparagine) residue and one more methionine residue per subunit than the ringspot st’rain. Differences of one residue per subunit were also found for threonine, alanine, and possibly leucine and tryptophan; the C strain yielded tsvo more residues of proline than the ringspot strain. With these five amino acids, however, the confidence limits overlap when compared and the small differences must be viewed with considerable caution. It thus appears that the two PVX strains in the present investigation are very similar in their amino acid compositions, a similarity that suggests a very close relationship betw-cen them. Studies of certain other plant viruses have also demonstrat,ed the presence of similar or ident,ical amino acid compositions between strains. No significant differences were found between t,wo biologically distinct isolates of tobacco mosaic virus (Knight, 1957) nor between three of tomato bushy stunt virus (de Fremery and Knight, 1955). Other strains of tobacco mosaic virus, of course, have significantly distinct amino acid compositions (Knight, 1947 ; Black and Knight, 1953). That the amino acid analyses of the C strain and ringspot strain proteins of PVX might yield very similar results was suggested by the identical complement-fixing capacities of these and other strains of PVX (Wright and Hardy, 1961) and by the similarity in type of symptom incited in tobacco plants. On the other hand, the
88
SHAW,
REICHMANN,
minor differences in symptoms described earlier were consistent enough to be considered significant and to suggest that the two isolates are distinct “strains.” The very small but statistically significant differences in composition mentioned above suggest that distinction between these two strains of PVX on the biological level is based on differences in chemical structure. REFERENCES BAUDART, E. (1957). Dosage des acides aminks du virus X de la pomme de terre par chromate Clectrorheophorese. Parasitica 13, 42-49. BAWDEN, F. C., and KLECZKOWSKI, A. (1948). Variations in the properties of potato virus X and their effects on its interactions with ribonuclease and proteolytic enzymes. J. Gen. Microbial. 2, 173-185. BLACK, F. L., and KNIGHT, C. A. (1953). A comparison of some mutants of tobacco mosaic virus. J. Biol. Chem. 202,51-57. CHIBNALL, A. C., MANGAN, J. L., and REES, M. W:. (1958). Studies on the amide and C-terminal residues in proteins. II. The ammonia nitrogen and amide nitrogen of various native protein preparations. Biochem. J. 68, 111-114. DE FREMERY, D., and KNIGHT, C. A. (1955). A chemical comparison of three strains of tomato bushy stunt virus. J. Biol. Chem. 214, 559-566. HILL, R. L., KIMMEL, J. R., and SNITH, E. I,. (1959). The structure of proteins. Ann. Rev. Biochem. 28,97-144. KLECZKOWSKI, A., and NIXON, H. L. (1950). An electron-microscope study of potato virus X in different stages of aggregation. J. Gen. Microbiol. 4, 220-224. KNIGHT, C. A. (1947). The nature of some of the chemical differences among strains of tobacco mosaic virus. J. Biol. Chem. 171, 297-308. KNIGHT, C. A. (1957). Some recent developments in the chemistry of virus mutants. Ciba Foundation Symposium on Nnture of Viruses, pp. 69-81. LADERERG,R. C., LARSON, R. H., and WALKER, J. C. (1950). Origin, interrelation and properties of ringspot strains of potato virus X in ilmerican potato varieties. Wisconsin Univ. Agr. Expt. Sta. Research Bull. No. 165,47 pp. MARKHAM, R., and SMITH, J. D. (1949). 9 source of error in amino-acid analysis. Nature 164,
1052.
AND
HATT
MOORE, S. (1961). Private communication. MOORE, S., and STEIN, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176, 367-388. MOORE, S., and STEIN, W. H. (1954). A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211,907-913. MOORE, S., SPACKWAS, D. H., and STEIN, W. H. (1958). Chromatography of amino acids on sulfonated polystyrene resins. Anal. Chem. 30, 11851190. NIU, C.-I., &ORE, V., and KNIGHT, C. A. (1958). The peptide chains of some plant viruses. Virology 6,22&-233. PATCHORNIK, A., LAWSON, W. B., and WITKOP, B. (1958). Selective cleavage of peptide bonds. II. The tryptophyl peptide bond and the cleavage of glucagon. J. Am. Chem. Sot. 80, 4747-4748. REES, M. W. (1946). The estimation of threonine and serine in proteins. Biochem. J. 40, 632-640. REICHMANN, M. E. (1959). Potato X virus. II. Preparation and properties of purified, non-aggregated virus from tobacco. Cm. J. Chem. 37, 4-10. REICHMANN, M. E. (1960). Degradation of potato virus X. J. Biol. Chem. 235, 2959-2963. REICHMANN, M. E., and HATT, D. L. (1961). The effect of the substitution of sulfhydryl groups on t,he macromolecular structure of potato virus X. Biochim. et Biophys. Acta 49,153-159. SCHRAM, E., MOORE, S., and BIGWOOD, E. J. (1954). Chromatographic determination of cystine as cysteic acid. Biochem. J. 57,33-37. SHAW, J. G., and LARSON, R. H. (1962). The amino acid composition of a strain of potato virus X. Phytopathology 52, 170-171. SMITH, E. L., and STOCKELL, A. (1954). Amino acid composition of crystalline carboxypeptidase. J. Biol. Chem. 207,501-514. SMITH, E. L., STOCKELL, A., and KIMMEL, J. R. (1954). Crystalline papain. III. Amino acid composition. J. Biol. Chem. 207, 551-561. SPACK~LIAN,D. H., STEIN, W. H., and MOORE, S. (1958). Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. WRIGHT, N. S., and HARDY, M. (1961). Fixation of complement by strains of potato virus X. Virology 13, 414-419.
18, 79-88 (1962)
Amino
Acid
Analyses
J. G. SHAW,l Research
Branch,
of the
Proteins Virus X
of Two
M. E. REICHMANN, Research
AND
May
of
Potato
D. L. HATT
Station, Canada Department Vancouver, B. C.?
Accepted
Strains
of Agriculture,
1, 1969
The amino acid compositions of the proteins isolated from two biologically similar strains of potato virus X have been determined by chromatography on columns of Amberlite IR-120. Independent estimations were made for tryptophan and cysteine, which was determined after oxidation to cysteic acid. The extent of variation of each amino acid was estimated by determining a confidence limit (P = 0.05) from the standard deviation of the mean. With both proteins, losses of threonine and serine and increases in valine, leucine, and isoleucine were observed as hydrolysis proceeded. The analyses indicate that both virus proteins (subunit molecular weight about 52,000) are composed of 463 amino acid residues of which about 40% consist of alanine, threonine, and aspartic acid (asparagine). The two virus proteins had remarkably similar amino acid compositions, the only statistically significant differences involving one aspartic acid (asparagine) and one methionine residue. INTRODUCTION
fate and resuspension in water followed by dialysis against water. This procedure favors aggregation of PVX particles and makes very difficult the removal of small amounts of nonvirus material (Bawden and Kleczkowski, 1948; Kleczkowski and Nixon, 1950). It seemed appropriate, therefore, to study the amino acids of this virus by analyzing hydrolyzates of the protein isolated by dissociation of highly purified, nonaggregated virus. A method for preparing nonaggregated PVX in a highly purified condition has been described in a previous report from this laboratory (Reichmann, 1959). More recently, the protein moiety of the virus has been isolated and found to have a subunit molecular weight of 52,000 * 2000 (Reichmann, 1960). An investigation of the amino acid contents of t,he proteins of two strains of PVX prepared by the same procedure forms the subject of this communication.
The amino acid content of potato virus X (PVS) has been the subject of two reports in recent years. Baudart (1957) investigated two European st’rains of this virus by paper chromatography and electrophoresis. More recently, Shaw and Larson (1962) presented the results of a preliminary study of the amino acid composition of another strain of PVX analyzed by chromatography on 8% cross-linked Amberlite IR-120 resin. In both cases, analyses were performed on hydrolyzates of preparations of the intact virus. Acid hydrolysis of nucleoprotein rather than simple protein results in the destruction of some amino acids and contributes to the formation of amino acid-like compounds during nucleic acid degradation (Markham and Smith, 1949). In addition, the analyses of Baudart (1957) were made on hydrolyzates of virus preparations purified by repeated precipitation with ammonium sul-
MATERIALS
1 Present address : Plant Research Institute, Central Experimental Farm, Ottawa, Ontario. ‘Contribution No. 40.
AND
METHODS
Viruses. Two strains of PVX, both apparently of the ringspot type, were prepared 79
80
SHAW, REICHMANN,
in quantities sufficient for amino acid analyses. One of them, strain C (Wright and Hardy, 1961), was isolated from potato in British Columbia, it is the same as that used in previous studies at this laboratory (Reichmann, 1959). It produces small, white, irregular rings on the inoculated leaves of Nicotiana tabacum L. (variety Haronova) , and rapidly becomes systemic, resulting in the formation of a severe mot,tle on the uninoculated leaves. The ot,her strain was the ringspot strain originally isolated from potato in Wisconsin (Ladeberg et al., 1950). It incites small, white, more regular rings on the inoculated leaves of tobacco and slowly becomes systemic, at which time a mild, irregular mottle appears on the upper leaves. To avoid compounding the already confusing situation in the nomenclature of PVX strains, the original designations-the C strain and the ringspot, strain -will be used throughout this report. Virus material was isolated from systemically infected tobacco plants grown in a room at 18°C with 120-160 foot-candles of fluorescent light for 16 hours per day. The symptoms mentioned above are those incited by each strain under these carefully controlled conditions. Virus samples were prepared by the method of Reichmann (1959)) which has consistently yielded highly purified, nonaggregated material. Amino acid analyses of virus proteins. Amino acid analyses were performed on hydrolyzates of t,he proteins isolated from both strains of the virus. Four different preparations of the C strain and three of the ringspot st.rain were used in the complete determinations. The protein was separated from the nucleic acid by treatment of the virus (in 0.005 M sodium citrate) with 2 M guanidine hydrochloride (Reichmann, 1960). After removal of the nucleic acid precipitate by centrifugation, the reagent was removed by dialysis against distilled water (brought to pH 7.5 with NaOH) at 2” for about 10 hours. Exhaustive dialysis was avoided since it usually led to severe aggregation of the protein. The dialyzed solution was centrifuged for 90 minutes in a refrigerated Spinco model L ultracentrifuge at 40,000 rpm; the small pellet, presumably incompletely split virus, was discarded. The
AND HATT
weight of protein in solution was determined by drying aliquots in a vacuum desiccator over PaOa and then at 107” to constant weight. Control solutions of guanidine hydrochloride in 0.005 M sodium citrate were dialyzed, centrifuged and weighed in the same manner. The small amounts of solute remaining in these solutions were subtracted from the weights of the appropriate protein samples. Presumably some ions interacted with and remained bound to the protein, but the error from this source was likely quite small. For hydrolysis, weighed aliquots of protein solution (about 10 mg) in Pyrex glass tubes were made 6 N in HCI by adding concentrated acid. The final volume was approximately 0.3 ml 6 il: HCl per milligram of protein. The tubes were sealed under vacuum and the samples were hydrolyzed for periods of 12, 24, and 48 hours in an oven at 107”. The use of different times of hydrolysis was deemed necessary because of evidence that amino acids differ considerably in their degree of stability and rates of release during hydrolysis (Hill et al., 1959). The hydrolyzates were light yellow in color; little or no insoluble humin was noticed. Excess HCl was removed by several cycles of evaporation in vacua at 40” and dilution in water, and the samples were finally made to volume in 0.2 M cit,rat.e buffer, pH 2.2 (Moore et al., 1958). Aliquots of the hydrolyzates equivalent to 1.5-2.0 mg protein were chromatographed on 15- and 150-cm columns of Amberlite IR-120 resin as described by Moore et al. (1958). Effluent fractions of 2.0 ml were collected with a volumetric fraction collector (Gilson Medical Electronics), a check on the weight of fractions being made at various intervals with tared tubes. The amino acid content of each fraction was determined by the photometric ninhydrin method of Moore and St.ein (1954) with the exception that reduced ninhydrin was formed by using stannous chloride (Moore and Stein, 1948) instead of by adding hydrindantin directly to the reagent mixture. The yield of each amino acid was determined by simple addit’ion of the color yields within each peak after subtraction of the appropriate baseline color. Carefully pre-
AMINO
ACID
ANALYSES
RESULTS
ASD
DISCUSSIOX
The analytical data from 12-, 24- and 48hour hydrolyzates of the two strain protcins are presented in Tables 1 and 2. Each analysis represents a single determination on the preparation indicated. The values obtained for all three periods of hydrolysis
TBBLE
1
(C STR.UN) AFTER DIFFERENT
-
81
PROTEISS
removal of excess performic acid (Moore, 1961). The excess SO, was removed with gentle swirling of the solution under partial vacuum to avoid severe foaming during subsequent evaporation to dryness. Aliquo ts of the hydrolyzate were chromatographed on 150-cm columns. The cysteic acid content was estimated by reference t’o the molar quantities of stable amino acids (aspartic acid, glutamic acid, and alanine) determined in the same analysis and for which the contents were known from analyses of unoxidized samples of the protein. The result,s were corrected for the 10% loss in recovery of cysteic acid.
pared solutions of known concentration of each amino acid (California Corporation for Biochemical Research) and the appropriate blanks were analyzed along with the effluent fractions in order to convert optical density readings to micromoles of amino acid. The recoveries of amino acids from freshly prepared columns were determined with the use of Spinco Calibration Mixture No. 1 (Beckman Instruments, Inc., Spinco Division) ; similar checks were made periodically during the course of the subsequent analysts. The tryptophan contents of the virus proteins were determined by ,V-bromosuccinimide cleavage of tryptophyl peptide bonds (Pat~chornik et al., 1958). Combined cyst& and cystcine determinations were made on separate samples of the proteins oxidized with performic arid prior to hydrolysis (Schram et aZ., 1954). At t#he completion of t’he oxidation reaction, sodium bisulfite was added to facilitate the
AMINO ACID REVOVERIES FROMPROTEIN OF PVX
OF PVX
TIMES OF HYDROLYSIS
Grams per 100 g protein Amino acid
I-
24 Hour
12 Hour
Average values f SD
48 Hour
I-
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine” Isoleucine Leucine Tyrosine Phenylalanme Lysine Histidine hrginine Ammonia
1
2
1
2
3
11.05 12.69 6.15 9.57 7.80 3.43 12.89 5.29 4.33 5.02 4.64 1.54 7.38
10.97 13.30 6.21 9.13
10.81 12.47 5.79 9.61 7.72 3.34 12.85 5.96 4.46 5.57 4.84
10.42 12.60 5.71 9.38 7.40 3.26 13.66 5.98 4.37 5.35 4.62 1.45 6.95 6.28 1.29 6.14 1.56
10.74 12.60 5.86 9.48 7.47 3.41 12.96 5.77 4.55 5.43 4.97 1.53 7.65 6.11 1.17 6.15 1.49
-
3.40 13.67 5.55 4.53 4.86 4.58 1.52 7.22
7.39 6.39 1.25 6.21 1.51
u Values obtained by extrapolation to zero time (see Fig. 1). h Yields corrected for 5!% loss (Moore et al., 1958). c 48.hour value used as average. d 24 and 48-hour values used in average.
1
2
6.38 I .21 6.23 1.80
10.87 11.56 5.30 9.48 7.i8 3.3-l 13.52 6.14 4.56 5.55 4.98 1.56 6.99 6.30 1.26 6.50 1.74
3
-
6.41 1.19 6.16 1.69
10.81 13.5oa 6.43a 9.44 i.63 3.36 13.26 6.14r 4.47 5.48 4.9@ 1.52 7.26 6.31 1.23 6.23 1.3w
zt 0.09
rt & f zk
0.07 0.08 0.03 0.16
f f
0.04 0.05d
III + zt f f
0.02 0.11 0.05 0.02 0.06
82
SHAW,
REICHMANN,
AND
TABLE
HATT
2
AMINO ACID RECOVERIES FROM PROTEIN OF PVX (RINGSPOT STRAIN) AFTER DIFFERENT TIMES OF HYDROLYSIS
-
-
Grams per 100 g protein
/ Amino acid
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine” Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Ammonia
12 Hour
11.23 13.29 6.26 9.40 7.34 3.41 14.03 4.66 3.97 5.04 4.71 1.37 7.00
24 Hour
11.31 13.20 6.21 9.25
11.00 12.57 5.80 9.66 7.38 3.49 13.22 5.85 4.10 5.48 4.82 1.49 7.34 6.27 1.14 6.33 1.50
7.22 3.63
13.79
-
-
5.15 4.12 4.82 4.56 1.41 7.23
-
11.28 12.56 6.12 9.19 7.15 3.28
13.09 5.28 4.23 5.61 4.60 1.38 7.33 6.35 1.21 6.35 1.58
48 Hour 2
2
1
11.20 12.58 6.04 9.44 7.21 3.18 13.24 5.94 4.12 5.47 4.49 1.53 7.34 6.33 1.21 6.41 1.44
11.78
11.07 11.45
12.93 6.04 9.25 6.89 3.34 13.16 5.90 4.37 5.59 5.20 6.39 1.21 6.25 1.55
11.43
5.56 9.16 7.15 3.58 13.51 6.21 4.20 5.38 5.13 1.40 7.55 6.47 1.21 6.10 1.84
-
1
--
5.56 9.43 6.87 3.49 13.45 6.15 4.21 5.41 5.05 1.40 6.98
-
-
4verage values f
SD
2 11.40 11.77 5.67 9.33 7.38 3.34
11.28 13.80a 6.43a 9.35 7.18 3.42 13.38 6.15 4.17 5.47 5.06 1.43 7.25 6.35 1.20 6.26 1.29=
12.89 6.09 5.38 5.01
6.29 1.23 6.10
1.64
zt 0.08
+ f f + + z!x rt f f f rt
0.05 0.06 0.05 0.12 0.03” 0.04 0.04d 0.04~ 0.02 0.08 0.03
f
0.01
f
0.05
-
a Values obtained by extrapolation to zero time (see Fig. 1). b Yields corrected for 5yo loss (Moore et al., 1958). c 48-hour values used in average. d 24. and 48.hour values used in average.
were included in the average when no significant increase or decrease with time was detected. The standard deviation of the mean (SZ) is included for each amino acid except threonine and serine (and, in the C strain, valine and leucine, for each of which only one value was used). With both proteins, the threonine and serine recovered decreased noticeably as time of hydrolysis increased. The concentrations of these amino acids were calculated by extrapolation of the values to zero time by the method of least squares, assuming a linear decrease of each amino acid with time (Fig. 1). After 24 hours’ hydrolysis, serine and threonine had decreased by 9.0% and 7.1%, respectively, in the C strain protein, and by 6.4% and 8.2%, respectively, in the ringspot strain protein. In neither case was
close. These different rates suggest the presence of adjacent residues in the proteins with differing degrees of susceptibility t,o hydrolytic attack. The values for methionine were corrected for the 5% loss reported by Moore et al. (1958). None of the other amino acids appeared to suffer destructive losses with prolonged hydrolysis. In both proteins, valine, leucine, and isoleucine yielded greater recoveries as the hydrolysis time increased. Isoleucine appeared to have been completely released after 24 hours and the values for the 24- and 48-hour hydrolyzates were used in the average. With valine and isoleucine, increases between 24 and 48 hours were obtained and only the 48-hour values were averaged. The increases in amounts of these three
the rate
drolysis are in accord with many previous observations on the relatively greater stability of the peptide bonds associated with
of decomposition
similar
between
strains though, especially in the case of serine, the extrapolated values were very
amino
acids
obtained
after
prolonged
hy-
AMINO
2 Sz 8cl4= t7
ACID ANALYSES
:.! SERINE
32-
.
1 0’
83
OF PVX PROTEISS
AMMONIA I
I
I
12
24
36
+-----+ IAMMONIA
I
I
I
48 0 12 TIME OF HYDROLYSIS (HOURS)
I
I
24
36
I 48
FIG. 1. Recovery of threonine, serine, and ammonia from acid hydrolyzates of potato virus X protein as a function of duration of hydrolysis. (A) C strain protein. (B) Ringspot strain protein. Lines represent the best fit to the data according to the method of least squares.
these amino acids (Hill et al., 1959). The recoveries of arginine appeared to increase as hydrolysis proceeded with the C strain and to decrease with the ringspot strain. These changes were slight, however, and all the values were included in the averages. Several analyses of unoxidized protein samples showed small, inconsistent peaks in the region of cystine, but, the highest of these amounted only to about 0.870 halfcystine. On t’he other hand, in samples oxidized with performic acid prior to hydrolysis the cysteic acid peak was well defined and separated from the other amino acid peaks. The molar yields of cysteic acid were determined by comparison with t,hose for aspartic acid, glutamic acid, and alanine in the same analyses. A mean value of 1.26 t 0.03 isi: = 0.03) g cysteine per 100 g protein was obtained for the C strain and agrees reasonably well with the result of Reichmann and Hatt (unpublished data) determined by the same procedure. Similar analyses on the ringspot strain yielded an average value of 1.12 i- 0.03 (sf = 0.02) g cysteine per 100 g protein. The lnain diffi-
culty in assessing the significance of these data arises from the necessity of analyzing large amounts of hydrolyzed protein in order to obtain cysteic acid peaks large enough to be properly evaluated. Under these circumstances, the color yields of the reference amino acids are very high and the baselines occasionally less steady than usual. This method of determining the cysteic acid content, however, proved to be more reproducible than that used for the other amino acids in unoxidized protein samples. In a previous study on the C strain of PVX, Reichmann and Hatt (1961) found 8 sulfhydryl groups per prot’ein subunit by means of p-chloromercuribenzoate substitution. Difficulties were encountered in the determinations because of the formation of emulsions with the dithizone-chloroform reagent. There is also the possibility that some nonspecific absorption of p-chloromercuribenzoate by the precipitated and denatured protein occurred. These factors may account for the discrepancy in cysteine determinations bet,ween this value and the data reported in the present investigation. On the
84
SHAW, REICHMANN,
other hand, the possibility of a more extensive destruction of cysteine during performic acid oxidation should be mentioned. In spite of this discrepancy, however, these investigations suggest that cysteine was the sole source of cysteic acid and that the virus protein contains no cystine. In terms of protein structure this would indicate that the subunit is a single polypeptide chain. Since tryptophan undergoes almost complete destruction during acid hydrolysis, the method of Patchornik et al. (1958)) involving the titration of C-tryptophyl peptide bonds cleaved by N-bromosuccinimide, was used. Average values of 3.45 (se = 0.07) and 3.34 (sz = 0.02) g/100 g virus protein were obtained for the C strain and ringspot strain, respectively, from analyses of three different preparations of each strain. The amide ammonia contents of the virus proteins were estimated by extrapolation of the values from 24- and 4%hour hydrolyzates to zero time by the method of least squares (Fig. 1). Values of 1.29% for the C strain and 1.30% for the ringspot strain were obtained. These values may exceed the actual amounts of ammonia liberated from amide groups during hydrolysis since Chibnall et al. (1958) found that various native proteins contain small amounts of free ammonia. Analyses of the virus proteins for free ammonia were not made, and the above estimates for amide ammonia must be regarded as maximum values only. In each case, the excess ammonia after 48 hours’ hydrolysis (calculated from the least squares values) was 0.026 mole/100 g protein. The amounts of threonine and serine destroyed during 48 hours hydrolysis were equivalent, to 0.027 mole for both proteins. As with other proteins (Rees, 1946; Smith and Stockell, 1954; Smith et al., 1954) it appears that the amount of ammonia liberated in excess of that at zero time of hydrolysis was due to the destruction of threonine and serine. The breakdown of cysteine and tryptophan apparently did not contribute to this excess ammonia. As reported by Moore et al. (1958)) analyses of standard calibration mixtures yielded recoveries of 100 f 3% for all the amino acids except methionine for which the recovery was 95%. Before a freshly poured
AND HATT
150-cm column would yield complete recoveries, however, it was necessary to pass alternating quantities of NaOH and citrate buffer (pH 3.25) through it six to eight times as in the regeneration cycle (Moore et al., 1958). The separation of peaks on the chromatograms was excellent except for threonineserine and tyrosine-phenylalanine. For the quantitative estimation of threonine and serine the color yield in the tube at the lowest point between the two peaks was divided equally between the two amino acids or in proportion to the yields in hhe tubes immediately preceding and following it. Since this amount constituted only a small percentage of the total color yields of each of the two peaks, the error involved, was probably slight. The tyrosine and phenylalanine determinations were made in the same manner. In the case of tyrosine, however, the error may be more appreciable because of its low content in the hydrolyzates. The total recoveries of amino acid residues from each strain protein were 96-97%, excluding the ammonia (Tables 3 and 4). Several factors may be responsible for this somewhat low recovery in weight. Small errors in the integration of the areas under the peaks of some amino acids are probably unavoidable as a result of variat,ions in the baseline. In general, the chromatoconsistently stable grams demonstrated baselines except in the valine-methionine region where a change in the pH of the eluent occurs. Because of the limited amounts of purified PVX that could be extracted from any group of plants, no more than about 10 mg of protein could be used in any single hydrolysis tube. With larger quantities of protein it is likely that the small losses inherent in the manipulations between hydrolysis and application of the samples to the columns would have been less significant. Some losses may also have been introduced during the manual procedure of ninhydrin color development. It is quite probable that the automatic recording method of amino acid analysis (Spackman et al., 1958) introduces less error in the determinations than does the manual method used in t’his study. Un-
.4MINO
ACID ANALYSES TABLE
Amino acid
COMPOSITION AND MOLECULAR
g Amino acid/lOOg protein”
g Amino cid residue/ OOgprotei; 1
10.81 13.50
9.35 11.46
6.43 9.44 7.63 3.36 13.26 6.14 4.47 5.48 4.98 1.52 7.26 6.31 1.23 6.23 3.45 1.26
5.33 8.28 6.44 2.55 10.58 5.20 3.93 4.73 4.30 1.37 6.47 5.53
-1.. Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alaninc Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Hist,idine Arginine Tryptophan” Cysteine c Ammonia
3
WEIGHT OF PVX
-
-
I
85
OF PVS PROTEINS
Minimal mol. wt.
(C STRAIN) -
Calculated no. Assumed residues (mol. I10. residues lalculated mol. wt. wt. = 51,140)’ -
1.30
1.09 5.59 3.15 1.07 1.22d
1,231
41.55 f 57.99 31.30
882 1,634
1,559 1,508 2,237 672
1,906 3,338 2,392 2,632 11,910 2,275 2,318 12,582 2,794 5,911 9,615 1,314
32.81 33.92 22.86 76.11 26.83 15.32 21.38 19.43 4.29 22.48 22.06 4.06 18.30 8.65 5.32 38.92
0.90
h 0.62 + 1.03 f 0.44 f 2.36 f f
0.35 0.65
f f f I!C zk f r!z
0.15 0.87 0.41 0.16 0.42 1.21 0.74
96.42
Totals
51,702 51,156
42 58 31 33 34 23
~
76
I
27 15 21 19 4 22 22 4 18 9 5 39d 463
50,654 51,447 51,272 51,451 51,072 51,462 50,070 50,232 50,008 47,640 50,050 50,996 50,328 50,292 53,199 48,075 51,246
/
(Av.
= 50,654)
a From Table 1. b Values of 3.46% (preparation 2), 3.577c (preparation 3), and 3.33@j, (preparation 4) obtained by method of Patchornik et al. (1958). Average = 3.45 (sz = 0.07) g tryptophan per 100 g protein. c Determined as cysteic acid in performic acid oxidized protein (Schram et al., 1954; Moore, 1961). Values ohtained by comparison to reference amino acids (see text) averaged to determine content,. d Not included in totals. e With tsi: at P = 0.05, n-l degrees of freedom. fortunately, only a single analysis of the C strain after 48 hours’ hydrolysis was obt,ained, thus the values for those amino acids increasing or decreasing as hydrolysis proceeded may be somewhat inaccurate for this strain. In addition, a period of hydrolysis longer than 48 hours might have yjclded further recoveries of valine and leucure and thus contributed to a more com-
plete analysis of each strain protein. The molecular weight of the protein subunit of the C strain of PVX has been established as 52,000 * 2000 from sedimentation-
diffusion
measurements
in this laboratory
(Reichmann, 1960). The discrepancies between this value and that of 74,000 calculated from C-terminal end-group analysis
by hydrazinolysis already
(Niu et cd., 1958) have
been discussed
(Reichmann,
1960).
Analysis of a sample of the ringspot strain protein in 2 M guanidine hydrochloride demonstrated a sedimentation rate in the Spinco model E analytical ultracentrifuge similar to that of the C strain. This suggested a subunit of similar or equal size. From the amino acid analyses, it was found that values slightly
lower than 52,000 better
fitted the data when considered as assumed molecular weights of the proteins. Thus, the values 51,140 and 51,200 for the C strain and ringspot
strain,
respectively,
were used
for calculating the number of residues prcsent in each subunit (Tables 3 and 4). On this basis, the general averages of 50,654 (C strain) and 50,999 (ringspot strain), which were obtained from t’he molecular weights calculated from all the amino acids,
86
SHAW,
REICHMANN,
AND
TABLE COMPOSITION
Amino acid
Aspsrtic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan” Cyst,eine c Ammonia Totals
AND MOLECULAR -
4
WEIGHT
g Amino acid/100 g proteina
g Amino cid residue I 00 g protein n
Minimal mol. wt.
11.28 13.80 6.43 9.35 7.18 3.42 13.38 6.15 4.17 5.47 5.06 1.43 7.25 6.35 1.20 6.26 3.34 1.12 1.29
9.75 11.71 5.33 8.21 6.06 2.60 10.67 5.20 3.67 4.72 4.37 1.29 6.46 5.57 1.06 5.61 3.05 0.95 1.21d
1,180 863 1,634 1,573 1,603 2,194 666 1,906 3,575 2,397 2,589 12,649 2,278 2,301 12,938 2,784 6,105 10,817 1,325
96.28
I
HATT
OF PVX
-
(RINGSPOT
Calculated no. residues (mol. wt. = 51,203)e 43.39 59.33 31.34 32.55 31.94 23.34 76.88 26.86 14.32 21.36 19.78 4.05 22.48 22.25 3.96 18.39 8.39 4.73 38.64
I
f
STRAIN)
Assumed I10. residues
0.76
lalculated
43 59 31 33 32 23 77 27 14 21 20 4 22 22 4 18 8 5 39d
rk 0.42 f 0.65 z!z 0.77 f 1.59 f 1.05 f 0.34 & 0.34 & 0.96 & 0.17 f 0.58 f 0.24 f 0.11 f 0.41 f 0.41 It 0.20
463
mol. wt.
50,740 50,917 50,654 51,909 51,296 50,462 51,282 51,462 50,050 50,337 51,780 50,596 50,116 50,622 51,752 50,112 48,840 54,085 51,675 (Av.
= 50,999)
-
a From Table 2. b Values of 3.30% (preparation I), 3.33% (preparation 2), and 3.38c/, (preparation 3) obtained by method of Patchornik et al. (1958). Average = 3.34 (sf = 0.02) g tryptophan per 100 g protein. c Determined as cysteic acid in performic acid oxidized protein (Schram et al., 1954; Moore, 1961). Values obtained bv comDarison to reference amino acids (see text) averaged to determine content. d Not included in totals. e With tsi: at I’ = 0.05, n-l degrees of freedom
are in close agreement with the molecular weight from physical measurements. The estimations of the number of residues of each amino acid in the two proteins are presented in Tables 3 and 4. From the % values (Tables 1 and 2) and n - 1 degrees of freedom, 95% confidence limits (ts, ) have been calculated. In the C strain protein, only one possible integer is within the calculated limits of error for glutamic acid, glycine, methionine, lysine, histidine, and arginine. Similarly, for proline, met,hionine, tyrosine, histidine, arginine, and tryptophan in the ringspot strain, only one possible integer is included within the 95% confidence intervals. An integral number of residues lying within the limits of error was not found for five amino acids (tyrosine in the C strain, and glutamic acid, isoleucine, lysine, and half-cystine in the ringspot
strain). Most of these, however, lie considerably closer to one integer than the other, and the nearest whole number has been assigned as the assumed number of residues per subunit. For the remaining amino acids in each strain, most of which are present in excess of 20 residues, there is more than one possible integer. High tsi values were obtained for several amino acids and consequently increased the number of possible integers. Proline gives a color with low extinction with ninhydrin reagent and analysis of this amino acid is somewhat less precise. Fewer data were available for the valine, isoleucine, and leucine calculations since the recoveries of these amino acids increased as hydrolysis proceeded. The independent determinations of cysteine and tryptophan were also based on fewer results than those of amino acids calcula,ted
AMINO
ACID
rlNALlrSES
after several hydrolysis times. Accurate intcgrat,ion of the aspartic acid and alanine peaks was more difficult because of the very large amounts of these amino acids present in the prot,eins. The analyses indicate that the PVX protein subunit is composed of 463 amino acid residues or slightly more when the 3-4s loss of recovery is considered. None of t,he common amino acids appear to be absent from either of the strain proteins examined nor do any occur to the extent of only one rcsiduc per subunit. Each strain contains less than 10 residues each of cysteine, tyrosine, tryptophan, and histidine per protein subunit. Of interest is the high content of alaninc, which comprises about 16.5% of the number of residues in each strain. Almost 40:/c of t,he number of residues in each protein is made up of the three amino acids alanine, threonine, and aspartic acid (asparagine). Based on the extrapolated ammonia values, each strain contains a maximum of 39 amide residues though, as mentioned earlier, some of these groups may represent free ammonia in the proteins. Chibnall et al. (1958) found errors in reported values for amide nitrogen in various proteins amounting to as much as 10% as a result. of the presence of free ammonia. With the possibility of this source of error in mind, it would appear that about half the aspartic and glutamic acid residues obtained after hydrolysis of the PVX strain proteins were originally in the amide form. These, however, cannot be assigned as definite numbers of asparaginyl or glutaminyl residues. The data of Shaw and Larson (1962) on another strain of PVX demonstrate a proportionately similar pattern in amino acid composition to those reported here though the percentage recoveries were somewhat less. It would be difficult, however, to compare the results of the two studies since the former involved a considerably less complete investigation of amino acid content. The results of the study by Baudart (1957) appear to be less similar to those reported herein but the different m&hods of preparation and analysis and the lower recoveries in the former make any comparisons unfeasible. In neither of the earlier reports were independent analyses made for cry-
OF PVX
PROTEINS
87
teine content, nor were different times of hydrolysis used to account fully for the labile and slowly released amino acids present. The analyses were made on hydrolyzates of intact virus and the higher glycinc yields obtained by Shaw and Larson (1962) can likely be attributed to the degradation of purines. It would seem, therefore, that the present analyses offer the most. complete data on the amino acid compositions of the PVX strains thus far examined. A comparison of the amino acid compositions of the C strain and ringspot strain proteins demonstrates a remarkable similarity between the two. Only in the cases of aspart’ic acid and methionine were differences significant at the 5% confidence level obtained. From the statistical analysis, the C strain would appear to contain one fewer aspartic acid (asparagine) residue and one more methionine residue per subunit than the ringspot st’rain. Differences of one residue per subunit were also found for threonine, alanine, and possibly leucine and tryptophan; the C strain yielded tsvo more residues of proline than the ringspot strain. With these five amino acids, however, the confidence limits overlap when compared and the small differences must be viewed with considerable caution. It thus appears that the two PVX strains in the present investigation are very similar in their amino acid compositions, a similarity that suggests a very close relationship betw-cen them. Studies of certain other plant viruses have also demonstrat,ed the presence of similar or ident,ical amino acid compositions between strains. No significant differences were found between t,wo biologically distinct isolates of tobacco mosaic virus (Knight, 1957) nor between three of tomato bushy stunt virus (de Fremery and Knight, 1955). Other strains of tobacco mosaic virus, of course, have significantly distinct amino acid compositions (Knight, 1947 ; Black and Knight, 1953). That the amino acid analyses of the C strain and ringspot strain proteins of PVX might yield very similar results was suggested by the identical complement-fixing capacities of these and other strains of PVX (Wright and Hardy, 1961) and by the similarity in type of symptom incited in tobacco plants. On the other hand, the
88
SHAW,
REICHMANN,
minor differences in symptoms described earlier were consistent enough to be considered significant and to suggest that the two isolates are distinct “strains.” The very small but statistically significant differences in composition mentioned above suggest that distinction between these two strains of PVX on the biological level is based on differences in chemical structure. REFERENCES BAUDART, E. (1957). Dosage des acides aminks du virus X de la pomme de terre par chromate Clectrorheophorese. Parasitica 13, 42-49. BAWDEN, F. C., and KLECZKOWSKI, A. (1948). Variations in the properties of potato virus X and their effects on its interactions with ribonuclease and proteolytic enzymes. J. Gen. Microbial. 2, 173-185. BLACK, F. L., and KNIGHT, C. A. (1953). A comparison of some mutants of tobacco mosaic virus. J. Biol. Chem. 202,51-57. CHIBNALL, A. C., MANGAN, J. L., and REES, M. W:. (1958). Studies on the amide and C-terminal residues in proteins. II. The ammonia nitrogen and amide nitrogen of various native protein preparations. Biochem. J. 68, 111-114. DE FREMERY, D., and KNIGHT, C. A. (1955). A chemical comparison of three strains of tomato bushy stunt virus. J. Biol. Chem. 214, 559-566. HILL, R. L., KIMMEL, J. R., and SNITH, E. I,. (1959). The structure of proteins. Ann. Rev. Biochem. 28,97-144. KLECZKOWSKI, A., and NIXON, H. L. (1950). An electron-microscope study of potato virus X in different stages of aggregation. J. Gen. Microbiol. 4, 220-224. KNIGHT, C. A. (1947). The nature of some of the chemical differences among strains of tobacco mosaic virus. J. Biol. Chem. 171, 297-308. KNIGHT, C. A. (1957). Some recent developments in the chemistry of virus mutants. Ciba Foundation Symposium on Nnture of Viruses, pp. 69-81. LADERERG,R. C., LARSON, R. H., and WALKER, J. C. (1950). Origin, interrelation and properties of ringspot strains of potato virus X in ilmerican potato varieties. Wisconsin Univ. Agr. Expt. Sta. Research Bull. No. 165,47 pp. MARKHAM, R., and SMITH, J. D. (1949). 9 source of error in amino-acid analysis. Nature 164,
1052.
AND
HATT
MOORE, S. (1961). Private communication. MOORE, S., and STEIN, W. H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176, 367-388. MOORE, S., and STEIN, W. H. (1954). A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211,907-913. MOORE, S., SPACKWAS, D. H., and STEIN, W. H. (1958). Chromatography of amino acids on sulfonated polystyrene resins. Anal. Chem. 30, 11851190. NIU, C.-I., &ORE, V., and KNIGHT, C. A. (1958). The peptide chains of some plant viruses. Virology 6,22&-233. PATCHORNIK, A., LAWSON, W. B., and WITKOP, B. (1958). Selective cleavage of peptide bonds. II. The tryptophyl peptide bond and the cleavage of glucagon. J. Am. Chem. Sot. 80, 4747-4748. REES, M. W. (1946). The estimation of threonine and serine in proteins. Biochem. J. 40, 632-640. REICHMANN, M. E. (1959). Potato X virus. II. Preparation and properties of purified, non-aggregated virus from tobacco. Cm. J. Chem. 37, 4-10. REICHMANN, M. E. (1960). Degradation of potato virus X. J. Biol. Chem. 235, 2959-2963. REICHMANN, M. E., and HATT, D. L. (1961). The effect of the substitution of sulfhydryl groups on t,he macromolecular structure of potato virus X. Biochim. et Biophys. Acta 49,153-159. SCHRAM, E., MOORE, S., and BIGWOOD, E. J. (1954). Chromatographic determination of cystine as cysteic acid. Biochem. J. 57,33-37. SHAW, J. G., and LARSON, R. H. (1962). The amino acid composition of a strain of potato virus X. Phytopathology 52, 170-171. SMITH, E. L., and STOCKELL, A. (1954). Amino acid composition of crystalline carboxypeptidase. J. Biol. Chem. 207,501-514. SMITH, E. L., STOCKELL, A., and KIMMEL, J. R. (1954). Crystalline papain. III. Amino acid composition. J. Biol. Chem. 207, 551-561. SPACK~LIAN,D. H., STEIN, W. H., and MOORE, S. (1958). Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. WRIGHT, N. S., and HARDY, M. (1961). Fixation of complement by strains of potato virus X. Virology 13, 414-419.