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Automated microsequence analysis in the presence of a synthetic “Carrier”
ANALYTICAL
BIOCHEMISTRY
Automated
60, 285-292 (1974)
Microsequence of a Synthetic JACK SILVER2
Division
Analysis “Carrier”l
AND
LEROY
in the
Presence
E. HOOD”
of Biology, California Institute of l’echnology, Pasadena, California 91109
Received December 24, 1973; accepted February
6, 1974
When small amounts of protein are subjected to automated sequence analysis, significant material washes out during the solvent wash steps and prevents extended analysis. Inclusion of a synthetic “carrier,” succinylated poly-ornithine, with the protein to be sequenced significantly reduces protein washout and permits extended automated microsequence analysis. This carrier also permits microquantities of protein to be sequenced in the presence of the detergent, sodium dodecyl sulfate.
Development of the automated Edman sequenator has rendered protein sequence analysis a routine procedure in many laboratories (1). Routine automated sequence analysis normally uses 200-500 nmoles of protein. The minimum amount of protein required for sequenceanalysis is limited by the sensitivity of the amino acid detection system and by the tendency of small quantities (i.e.,
AND METHODS
Automated Sequence Analyses Protein dissolved in 200-300 pliters of water was placed in a Beckman model 890 sequencer, dried under vacuum, and examined using a program similar to that described previously which uses“Quadrol” as the coupling buffer (2). Phenylthiohydantoin (PTH) amino acids were hydrolyzed to free amino acids with HI (3) and analyzed by means of a Durrum ’ This work was supported by GM 06965, NSF (GB 276051, NIH (AI 10781). ’ Recipient of National Institutes of Health Postdoctoral Fellowship (AI 10781). 3 Recipient of Career Development Award (AI 20388). 285 Copyright,@ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
286
SILVER
AND
HOOD
D500 amino acid analyzer. When protein was to be sequenced in the presence of “carrier,” 5-7 mg of succinylated polyornithine dissolved in 200 pliters of water were placed in the sequenator cup and subjected to two complete sequence cycles. Protein samples (50-100 pg) were then dissolved in 200 pliters of water, added to the cup, and dried under vacuum prior to commencement of the sequencing cycle. Preparation
of Succinylated
Poly-Ornithine
Poly-L-ornithine HBR (Research Plus Laboratories, Inc., 200 mg) and Triema HCl (15.8 mg) were dissolved in a minimum amount of water and brought to pH 9.5 by the addition of 3 N NaOH. Finely ground succinic anhydride (4.0 g) was sIowly added to the sohrtion while the pH was maintained at 9.5 by the automatic addition of 3 N NaOH. The solution was then dialyzed against distilled water for 2 days with four or five changes of water and subsequently lyophilized. RESULTS
AND
DISCUSSION
Samples of chicken egg-white lysozyme were examined in an automated sequenator in the presence and absence of succinylated polyornithine. In order to facilitate interpretation of the following data a brief description of the sequential steps in a typical sequence cycle is presented. [A more detaiIed description may be found elsewhere (4).] The protein to be sequenced is loaded into the sequenator chamber and coupled to phenylisothiocyanate in the, presence of a tertiary amine buffer. At the conclusion of the coupling reaction the buffer is extracted with ethyl acetate. Cyclization and cleavage to the resulting thiazolinone derivative of the N-terminal amino acid is effected by a strong organic acid under anhydrous conditions. This derivative is then extracted with butyl chloride while the insoluble. shortened protein remains in the sequenator cup and is subjected to additional degradation cycles. The extracted thiazolinone derivative is converted to the phenylthiohydantoin derivative by aqueous dilute acid and extracted with ethyl acetate. Identification of the PTH-amino acid may be accomplished by thin-layer chromatography, gas-liquid chromatography, or hydrolysis of the PTH derivative to its component amino acid and subsequent identification on an amino acid analyzer. After the PTH-amino acid from each step has been hydrolyzed and analyzed on an amino acid analyzer, the data for each step are converted to amino acid mole percent. The resulting data for each step are a combination of a residual background superimposed on that of the actual amino acid residue at the particular step. Several factors place severe Iimitations on the number of degradation
AUTOMATED
287
MICROSEQUENCING
cycles which a protein may be subjected to and successfully analyzed. First, small, though significant, amounts of protein are extracted during the ethyl acetate and butyl chloride wash steps. This results in reduced yields during subsequent cycles. Some of this protein is undoubtedly also extracted along with the PTH-amino acids from the aqueous phase into the organic phase prior to acid hydrolysis and contributes to the initial and subsequent amino acid background. Second, during each cleavage step of the cycle, a small amount of nonspecific cleavage of peptide bonds occurs, presumably an acidolysis caused by the organic acid. Each gratuitous cleavage results in creation of a new free S-terminus, and a new Amino
asp du pro d) ala ABA val ileu leu tYr phe lys Tot,al ~--
in
nmoles sample
asP glu Pro t%lY ala ABA val ileu leu tyr phe lYS
Acid
TABLE 1 of Seuuenat80r Run in the Presence
Analvsis
of “Carrier”~~
lys Ib
val 2
phe 3
dY 4
arg <5
cys 6
glu 7
leu S
ala 9
ala 10
9.G 11.6 2.6 15.6 12.2 1.0 4.9 3.5 6.3 2.2 3.0 2_2_7
7.9 8.2 1.0 16.8 9.7 0.5 39.7 2.7 4.6 2.4 2.7 3.9
6.8 6.4 1.2 13.6 9.8 0.7 5.1 2.0 3.0 2.2 46.6 2.7
9.2 9.0 3.6 40.2 11.4 0.8 4.5 3.1 5.6 1.8 6.4 4.4
11.0 10.0 5.3 32.9 14.3 1.9 4.9 3 .3 5.0 2.2 3.7 5.5
9.4 8.9 4.5 17.0 28.3 3.0 5.5 1.8 7.0 4.9 4.6 5.0 ___-
7.6 32.3 3.3 12.7 17.2 3.0 4.6 1.7 t5.4 4.8 3.0 4.4 .-.
6.7 18.9 3.0 10.6 10.9 1.7 3.2 1.6 34.8 1.9 2.1 4.7
8.0 11.7 3.0 14.7 32.8 1.1 3 .5 1.9 15.x 1 .9 2.1 3.5
7.0 8.4 3 7 10.5 41.7 2.7 3.4 1.4 s.1 3 6 3.4 4.4
3.4d
3.1
3.7
5 2
2.6
3.4
3.7
3.2
4.5
3.7
ala 11
met 12
1YS 13
arg 14
his 15
dY 16
leu 17 ___-.
asp 1X -
asn 19
tyr 20
7.1 6.0 1.5 10.6 @.CJ 1.6 3.0 2.0 5.4 1.8 2.0 3.2
9.5 9.2 3.5 13.4 33.3 3.8 4.8 1.6 8.3 5.0 3.2 4.6
9.4 9.8 4.6 16.6 24.8 3.1 4.7 2.1 7.4 4.9 3.1 9A
10.4 8.9 5.6 14.5 23.7 4.6 5.8 1.5 7.7 6.0 3.8 7.3
9.8 9.0 6.0 16.7 22.6 4.9 5.2 2.0 7.8 5.5 4.1 6.5
11.0 10.4 0.0 33.3 19.3 2.6 5.0 2.9 5.9 2.2 3.3 4.2
9.3 6.0 2.9 21.7 22.0 4.0 5.0 1.6 15.7 4.5 3.0 4.4
m 8.5 2.4 17.8 19.6 3 .9 4.9 1.6 10.6 5.1 3.3 5.2
21.5 11.0 2.8 18.X 17.8 2.3 4.8 3.4 9.5 2.1 2.9 3.1
17.2 11.3 3 1 19.5 18.9 2.5 4 3 :3. 1 7.1 7.1 2.6 3.4
(Continued)
288
SILVJXEt
TABLE
=P !a pro @Y
ala ABA val ileu leu tyr he lYS
AND
HOOD
1 (Continued)
arg
dY
tyr
21
22
23
ser 24
leu 25
12.1 12.0 12.0 9.5 2.6 4.0 23>23.5 20.1 17.6 2.2 2.9 4.9 4.6 3.8 3.2 7.2 5.7 4.4 10.6 3.6 2.7 4.0 4.1
11.9 8.7 1.4 24.7 23.8 2.2 4.7 2.9 5.1 7.6 2.8 4.2
11.3 9.8 2.7 21.8 21.7 2.5 5.0 3.4 10.5 4.4 3.1 3.9
18.9 8.9 2.6 18.3 18.3 2.2 5.5 3.2 6.9 8.9 2.7 3.5
a Lysozyme (100 pg, 7 nm) was sequenced in the presence of succinylated polyornithine (5 mg). PTH-amino acids were extracted with ethyl acetate, hydrolyzed, and analyzed on an amino acid analyzer. The aqueous partitioning material was not analyzed, precluding identification of arginine and histidine residues. PTH-cysteine a.nd PTHserine are degraded to alanine, PTH-asparagine is converted to aspartic acid, and methionine is destroyed by the acid hydrolysis procedure. The correct sequence is presented above the step number while the sequence deduced from the data is underlined. b Step number. c MoIe percent of given step. d Corrected against an internal standard of PTH-norleucine (2). initiation site for the Edman degradation cycle. The result is a background of PTH-amino acids which approximates the amino acid composition of the protein. With each succeeding cycle additional N-termini are created resulting in a continuously increasing background. Finally, incomplete Edman reactions lead to sequence overlap in which the Nterminal residue for the preceding step is cleaved a cycle late. These and other factors limit the successive yields at each cycle (repetitive yields) such that the yield of actual N-terminal amino acids approaches the background level, and accordingly, the data eventuaIly become uninterpretable. Therefore the actual amino acid at a particular step may be deduced from a sharp increase in its respective mole percent value and a gradual decline during subsequent cycles due to sequence overlap. The effect of “carrier” on the amount of protein washout is reflected in the total nanomoles of all amino acids present at each step in the presence and absence of “carrier” (Tables 1 and 2). The presence of “carrier” permitted the yield observed at step 1 of 3 nanomoles to remain fairly constant throughout the first ten steps (Table 1). In contrast, in the absence of “carrier,” a high initial yield (15.1 nmoles) decreased rapidly to level
AUTOMATED
Amino
Acid
Analysis
TABLE 2 of Sequenator Run in the Absence 1YS
1 =P du Pro dY ala
ABA val ileu leu tYr phe b Total
nmoles
in sample
289
MICROSEQUENCINC
val 2
phe 3
dY
12.0
4
of “Carrier”n cys 6b
glu
leu
7
8
9.1 22.2 4.5 22.2 1-i .5 0.8 5.2 3 .2
10.7 8.8 7.7 19.1 14.1 1.6 5.1 5.0 6.7 3.5 3.9 13.8
6.8 3.2 24.2 13.8 0.6 16.2 5.1 5.9 3.9 3.6 4.7
10.7 8.5 1.5 20.3 12.1 1.6 6.2 4.0 5.6 3.7 2o.Fj 5.3
11.6 8.7 5.3 28.0 12.7 1 .o 6.1 4.3 5.5 5.4 6.1 5.4
16.2 8.1 6.1 22.4 16.5 6.4 4.9 4.7 3 .8 4.2 6.2
10.3 22.Fi 1.7 28.3 13.0 0.9 4.7 3.2 4.3 3.1 3 5 4.5
15.4c
6.3
4.2
3 .3
2.3
2.9
D Lysozyme (100 II@;) was sequenced and analyzed in described in Table 1 except for the omission of succinylated b Step 5 was inadvertently lost. c Corrected against an internal standard of PTH-norleucine.
a
0.0
manner identical
7.4 2. 8 3 1 4.0 5 2 to that
poly-ornithine.
off at about 3 nmoles (Table 2). Accordingly, the addition of “carrier” permitted the sequential degradation and identification of 19 of 25 amino acid residues from the N-terminus of lysozyme using as little as 7 nmoles of protein (ca. l/30 the amount normally used) (Table 1). In contrast, in the absence of “carrier,” protein loss during the sequence cycles made amino acid identification difficult after step 7 (Table 2). This is best illustrated by comparing the mole percent values of leucine at steps 7 and 8 (cf. Tables 1 and 2). In the presence of “carrier,” the mole percent of leucine rises from 5.4 to 34.8 (544% increase) while in its absence these values change from 4.3 to 7.4 (727 o increase). The ability to assign an amino acid residue to a particular step is a function of the change in amino acid mole percent during consecutive steps. The inclusion of succinylated poly-ornithine has a profound effect on this value and concomitantly the ability to sequence lysozyme further. Comparable results were obtained using even smaller amounts of lysozyme (50 /~g, 3 nm) in the presence of “carrier.” Plots of amino acid yields indicate that these differences are due to a drastic decrease in the repetitive yield when “carrier” is omitted, presumably due to protein washout during the sequence cycle (Fig. 1). The repetitive yield in the presence of succinylated poly-ornithine calculated from the yields of glycine at step 4 and 16, and from the yields of leucine
290
SILVER
AND
HOOD
FIG. 1. Amino acid yields of lysozyme sequenator runs in the presence (0) and absence (0) of succinylated poly-ornithine. Background values were subtracted by comparison to a previous step. LORW due to manipulation were corrected against an internal standard of PTH-norleucine.
at steps 8 and 25 are 92 and 91%, respectively (Fig. 1). This compares favorably with sequence runs using macroamounts of protein. Omission of “carrier” reduces the repetitive yield to approximately 67%. Since several groups of proteins of significant interest (e.g., mouse H-2 alloantigens, nonhistone chromosomal proteins) require sodium dodecyl sulfate (SDS) for solubilization and subsequent purification (5,6), it was of interest to determine the feasibility of automated microsequence analysis in the presence of SDS. Light chain from homogeneous rabbit antibody of known sequence was used to determine the effect of succinylated poly-ornithine on the automated sequence analysis of protein in the presence of SDS. “Carrier” permitted the facile identification of the first six amino acid residues, while its omission made identification difficult past step 2 (Table 3). Weiner et al. (7) using a manual dansyl-Edman procedure were able to sequence microquantities of protein in the presence of SDS. Our results indicate that automated microprotein sequence analysis in the presence of SDS is also possible. The choice of succinylated poly-ornithine as putative “carrier” was based on several considerations. Ornithine, a homolog of lysine, is readily resolved from the other basic amino acids on an amino acid analyzer. Since it is not a naturally occurring amino acid its presence on amino
AUTOMATED
Automated
291
MICROSEQUENCING
Microsequence
TABLE 3 Analysis in the Presence of SDS0
A asP 1 SP
pro 2
val
leu
thr
3
4
5
glu 6
asp
Pro
val
leu
1
2
3
4
9.6
7.5
7.7
8.3
7.1
20.“ 1
10.5
9.s
8.4
!a
5.6
7.5
7.9
X.1
x.5
18.7
12.5
10.9
12.6
11.5
pro @Y
2.7 9.0
17.1 8.9
7.8 11.9
s .7 10.2
4.4 10.7
3 0 11.0
3.2 9.6
gg 11.2
5.5 11.3
7.4 14.6
ala
19.1 5.1
25.2 7.5
20.5 7.1
19.2 7.3
19.9 20.2
17.5 10.2
18.6 :3 .3
20.7 4.9
20.7 4.5
20.3 5.4
3.7 1.6
5.7 2.2
23.1 1.4
9,s 1.6
6.0 2.0
10.1 4.0
6.8 2.9
4.1
7.2
6.1
25.))
14.0
phe
2.0 1.8
3.4 3.0
2.4 2.7
2.6 2.5
2.7 2.. 5
2.:~ 2 I
1YS
‘2 3
‘2 7
1.7
1.6
1.9
5.7
8.1
6.8
7.4
6.4
ABA” val ileu leu t,yr
Total nmoles in sample
42.9
B
13.1
5.2 6 3
6.6 3.6
6.6 3 2
9 0
9 .2
9.7
3.7 s .!I
3 3 3 7
3 .!I 3 7
2 s 2.5
1.7
4,s
2.3
4.4
4.0
6.1
21 .!I
7.7
11.5
14.9
4 s
a Homogeneous rabbit light chain (100 rg) was dissolved in 200 Jiters of 0.5oj, SDS and sequenced in the presence of 5 mg of succinylated poly-ornithine (A) and in its absence (B). b PTH-threonine is degraded t#oa-amino butlyric acid (ABA) during the acid hydrolysis procedure. c Corrected against an internal standard of PTH-norleucine.
acid chromatograms is not confusing. The random polymer of ornithine was succinylated in order to render it highly anionic and soluble during
alkaline conditions (coupling with phenylisothiocyanate) and insoluble during the ethyl acetate and benzene washes during the sequenator cycles. These are ideal properties for any polypeptide undergoing the three cycle Edman reaction. The inclusion of “carrier” with the protein to be sequenced significantly limits protein washout and permits extended automated microsequence analysis. Recently, the development of a new reagent, “fluorescamine,” for amino acid detection permits a 50- to lOO-fold increase in sensitivity (8). This reagent used in conjunction with our synthetic “carrier” should permit. extended automated sequence analysis at the picomole level. Note: Preliminary results indicate that significantly less protein washout occurs when the N,N-Dimethylbenzylamine (DMBA) program is used (9) even when carrier is omitted.
292
SILVER
AND
HOOD
REFERENCES 1. EDMAN, P., AND BEGG, G. (1967) EUT. J. Biochem. 1, SO. 2. HOOD, L,, MCKEAN, D., FARNSWORTH, V., AND POTPER, M. (1973) Biochemistry 12, 741. 3. SMITHIES, O., GIBSON, D. M., FANNING, E. M., GOODFLIESH,R. M., GILMAN, J. G., AND BALLANTYNE, D. L. (1971) Biochemistry 10, 4912. 4. NEEDLEMAN, S. B. (Ed.) (1970) Protein Sequence Determination, p. 229, SpringerVerlag, New York. 5. WV, F., ELQIN, S., AND HOOD, L. (1973) Biochemistry 12, 2792. 6. SCHWARTZ, B. D., KATO, K., CULLEN, S. E., AND NATHENSON, S. G. (1973) Biochemistry 12, 2157. 7. WEINER, A. M., PLATT, T., AND WEBER, K. (1972) J. BioE. Chem. 246, 3242. 8. UDENFRIEND, S., STEIN, S., BOHLEN, P., DAIRMAN, W., LEIMGRIBER, W., AND WEIGELE, M. (1972) Science 178, 871. 9. HERMODSON, M. A., ERICSSON, L. H., TITANI, K., NEURATH, H., AND WALSH, K. A. ( 1972) Biochemistry 11, 4493,
BIOCHEMISTRY
Automated
60, 285-292 (1974)
Microsequence of a Synthetic JACK SILVER2
Division
Analysis “Carrier”l
AND
LEROY
in the
Presence
E. HOOD”
of Biology, California Institute of l’echnology, Pasadena, California 91109
Received December 24, 1973; accepted February
6, 1974
When small amounts of protein are subjected to automated sequence analysis, significant material washes out during the solvent wash steps and prevents extended analysis. Inclusion of a synthetic “carrier,” succinylated poly-ornithine, with the protein to be sequenced significantly reduces protein washout and permits extended automated microsequence analysis. This carrier also permits microquantities of protein to be sequenced in the presence of the detergent, sodium dodecyl sulfate.
Development of the automated Edman sequenator has rendered protein sequence analysis a routine procedure in many laboratories (1). Routine automated sequence analysis normally uses 200-500 nmoles of protein. The minimum amount of protein required for sequenceanalysis is limited by the sensitivity of the amino acid detection system and by the tendency of small quantities (i.e.,
AND METHODS
Automated Sequence Analyses Protein dissolved in 200-300 pliters of water was placed in a Beckman model 890 sequencer, dried under vacuum, and examined using a program similar to that described previously which uses“Quadrol” as the coupling buffer (2). Phenylthiohydantoin (PTH) amino acids were hydrolyzed to free amino acids with HI (3) and analyzed by means of a Durrum ’ This work was supported by GM 06965, NSF (GB 276051, NIH (AI 10781). ’ Recipient of National Institutes of Health Postdoctoral Fellowship (AI 10781). 3 Recipient of Career Development Award (AI 20388). 285 Copyright,@ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
286
SILVER
AND
HOOD
D500 amino acid analyzer. When protein was to be sequenced in the presence of “carrier,” 5-7 mg of succinylated polyornithine dissolved in 200 pliters of water were placed in the sequenator cup and subjected to two complete sequence cycles. Protein samples (50-100 pg) were then dissolved in 200 pliters of water, added to the cup, and dried under vacuum prior to commencement of the sequencing cycle. Preparation
of Succinylated
Poly-Ornithine
Poly-L-ornithine HBR (Research Plus Laboratories, Inc., 200 mg) and Triema HCl (15.8 mg) were dissolved in a minimum amount of water and brought to pH 9.5 by the addition of 3 N NaOH. Finely ground succinic anhydride (4.0 g) was sIowly added to the sohrtion while the pH was maintained at 9.5 by the automatic addition of 3 N NaOH. The solution was then dialyzed against distilled water for 2 days with four or five changes of water and subsequently lyophilized. RESULTS
AND
DISCUSSION
Samples of chicken egg-white lysozyme were examined in an automated sequenator in the presence and absence of succinylated polyornithine. In order to facilitate interpretation of the following data a brief description of the sequential steps in a typical sequence cycle is presented. [A more detaiIed description may be found elsewhere (4).] The protein to be sequenced is loaded into the sequenator chamber and coupled to phenylisothiocyanate in the, presence of a tertiary amine buffer. At the conclusion of the coupling reaction the buffer is extracted with ethyl acetate. Cyclization and cleavage to the resulting thiazolinone derivative of the N-terminal amino acid is effected by a strong organic acid under anhydrous conditions. This derivative is then extracted with butyl chloride while the insoluble. shortened protein remains in the sequenator cup and is subjected to additional degradation cycles. The extracted thiazolinone derivative is converted to the phenylthiohydantoin derivative by aqueous dilute acid and extracted with ethyl acetate. Identification of the PTH-amino acid may be accomplished by thin-layer chromatography, gas-liquid chromatography, or hydrolysis of the PTH derivative to its component amino acid and subsequent identification on an amino acid analyzer. After the PTH-amino acid from each step has been hydrolyzed and analyzed on an amino acid analyzer, the data for each step are converted to amino acid mole percent. The resulting data for each step are a combination of a residual background superimposed on that of the actual amino acid residue at the particular step. Several factors place severe Iimitations on the number of degradation
AUTOMATED
287
MICROSEQUENCING
cycles which a protein may be subjected to and successfully analyzed. First, small, though significant, amounts of protein are extracted during the ethyl acetate and butyl chloride wash steps. This results in reduced yields during subsequent cycles. Some of this protein is undoubtedly also extracted along with the PTH-amino acids from the aqueous phase into the organic phase prior to acid hydrolysis and contributes to the initial and subsequent amino acid background. Second, during each cleavage step of the cycle, a small amount of nonspecific cleavage of peptide bonds occurs, presumably an acidolysis caused by the organic acid. Each gratuitous cleavage results in creation of a new free S-terminus, and a new Amino
asp du pro d) ala ABA val ileu leu tYr phe lys Tot,al ~--
in
nmoles sample
asP glu Pro t%lY ala ABA val ileu leu tyr phe lYS
Acid
TABLE 1 of Seuuenat80r Run in the Presence
Analvsis
of “Carrier”~~
lys Ib
val 2
phe 3
dY 4
arg <5
cys 6
glu 7
leu S
ala 9
ala 10
9.G 11.6 2.6 15.6 12.2 1.0 4.9 3.5 6.3 2.2 3.0 2_2_7
7.9 8.2 1.0 16.8 9.7 0.5 39.7 2.7 4.6 2.4 2.7 3.9
6.8 6.4 1.2 13.6 9.8 0.7 5.1 2.0 3.0 2.2 46.6 2.7
9.2 9.0 3.6 40.2 11.4 0.8 4.5 3.1 5.6 1.8 6.4 4.4
11.0 10.0 5.3 32.9 14.3 1.9 4.9 3 .3 5.0 2.2 3.7 5.5
9.4 8.9 4.5 17.0 28.3 3.0 5.5 1.8 7.0 4.9 4.6 5.0 ___-
7.6 32.3 3.3 12.7 17.2 3.0 4.6 1.7 t5.4 4.8 3.0 4.4 .-.
6.7 18.9 3.0 10.6 10.9 1.7 3.2 1.6 34.8 1.9 2.1 4.7
8.0 11.7 3.0 14.7 32.8 1.1 3 .5 1.9 15.x 1 .9 2.1 3.5
7.0 8.4 3 7 10.5 41.7 2.7 3.4 1.4 s.1 3 6 3.4 4.4
3.4d
3.1
3.7
5 2
2.6
3.4
3.7
3.2
4.5
3.7
ala 11
met 12
1YS 13
arg 14
his 15
dY 16
leu 17 ___-.
asp 1X -
asn 19
tyr 20
7.1 6.0 1.5 10.6 @.CJ 1.6 3.0 2.0 5.4 1.8 2.0 3.2
9.5 9.2 3.5 13.4 33.3 3.8 4.8 1.6 8.3 5.0 3.2 4.6
9.4 9.8 4.6 16.6 24.8 3.1 4.7 2.1 7.4 4.9 3.1 9A
10.4 8.9 5.6 14.5 23.7 4.6 5.8 1.5 7.7 6.0 3.8 7.3
9.8 9.0 6.0 16.7 22.6 4.9 5.2 2.0 7.8 5.5 4.1 6.5
11.0 10.4 0.0 33.3 19.3 2.6 5.0 2.9 5.9 2.2 3.3 4.2
9.3 6.0 2.9 21.7 22.0 4.0 5.0 1.6 15.7 4.5 3.0 4.4
m 8.5 2.4 17.8 19.6 3 .9 4.9 1.6 10.6 5.1 3.3 5.2
21.5 11.0 2.8 18.X 17.8 2.3 4.8 3.4 9.5 2.1 2.9 3.1
17.2 11.3 3 1 19.5 18.9 2.5 4 3 :3. 1 7.1 7.1 2.6 3.4
(Continued)
288
SILVJXEt
TABLE
=P !a pro @Y
ala ABA val ileu leu tyr he lYS
AND
HOOD
1 (Continued)
arg
dY
tyr
21
22
23
ser 24
leu 25
12.1 12.0 12.0 9.5 2.6 4.0 23>23.5 20.1 17.6 2.2 2.9 4.9 4.6 3.8 3.2 7.2 5.7 4.4 10.6 3.6 2.7 4.0 4.1
11.9 8.7 1.4 24.7 23.8 2.2 4.7 2.9 5.1 7.6 2.8 4.2
11.3 9.8 2.7 21.8 21.7 2.5 5.0 3.4 10.5 4.4 3.1 3.9
18.9 8.9 2.6 18.3 18.3 2.2 5.5 3.2 6.9 8.9 2.7 3.5
a Lysozyme (100 pg, 7 nm) was sequenced in the presence of succinylated polyornithine (5 mg). PTH-amino acids were extracted with ethyl acetate, hydrolyzed, and analyzed on an amino acid analyzer. The aqueous partitioning material was not analyzed, precluding identification of arginine and histidine residues. PTH-cysteine a.nd PTHserine are degraded to alanine, PTH-asparagine is converted to aspartic acid, and methionine is destroyed by the acid hydrolysis procedure. The correct sequence is presented above the step number while the sequence deduced from the data is underlined. b Step number. c MoIe percent of given step. d Corrected against an internal standard of PTH-norleucine (2). initiation site for the Edman degradation cycle. The result is a background of PTH-amino acids which approximates the amino acid composition of the protein. With each succeeding cycle additional N-termini are created resulting in a continuously increasing background. Finally, incomplete Edman reactions lead to sequence overlap in which the Nterminal residue for the preceding step is cleaved a cycle late. These and other factors limit the successive yields at each cycle (repetitive yields) such that the yield of actual N-terminal amino acids approaches the background level, and accordingly, the data eventuaIly become uninterpretable. Therefore the actual amino acid at a particular step may be deduced from a sharp increase in its respective mole percent value and a gradual decline during subsequent cycles due to sequence overlap. The effect of “carrier” on the amount of protein washout is reflected in the total nanomoles of all amino acids present at each step in the presence and absence of “carrier” (Tables 1 and 2). The presence of “carrier” permitted the yield observed at step 1 of 3 nanomoles to remain fairly constant throughout the first ten steps (Table 1). In contrast, in the absence of “carrier,” a high initial yield (15.1 nmoles) decreased rapidly to level
AUTOMATED
Amino
Acid
Analysis
TABLE 2 of Sequenator Run in the Absence 1YS
1 =P du Pro dY ala
ABA val ileu leu tYr phe b Total
nmoles
in sample
289
MICROSEQUENCINC
val 2
phe 3
dY
12.0
4
of “Carrier”n cys 6b
glu
leu
7
8
9.1 22.2 4.5 22.2 1-i .5 0.8 5.2 3 .2
10.7 8.8 7.7 19.1 14.1 1.6 5.1 5.0 6.7 3.5 3.9 13.8
6.8 3.2 24.2 13.8 0.6 16.2 5.1 5.9 3.9 3.6 4.7
10.7 8.5 1.5 20.3 12.1 1.6 6.2 4.0 5.6 3.7 2o.Fj 5.3
11.6 8.7 5.3 28.0 12.7 1 .o 6.1 4.3 5.5 5.4 6.1 5.4
16.2 8.1 6.1 22.4 16.5 6.4 4.9 4.7 3 .8 4.2 6.2
10.3 22.Fi 1.7 28.3 13.0 0.9 4.7 3.2 4.3 3.1 3 5 4.5
15.4c
6.3
4.2
3 .3
2.3
2.9
D Lysozyme (100 II@;) was sequenced and analyzed in described in Table 1 except for the omission of succinylated b Step 5 was inadvertently lost. c Corrected against an internal standard of PTH-norleucine.
a
0.0
manner identical
7.4 2. 8 3 1 4.0 5 2 to that
poly-ornithine.
off at about 3 nmoles (Table 2). Accordingly, the addition of “carrier” permitted the sequential degradation and identification of 19 of 25 amino acid residues from the N-terminus of lysozyme using as little as 7 nmoles of protein (ca. l/30 the amount normally used) (Table 1). In contrast, in the absence of “carrier,” protein loss during the sequence cycles made amino acid identification difficult after step 7 (Table 2). This is best illustrated by comparing the mole percent values of leucine at steps 7 and 8 (cf. Tables 1 and 2). In the presence of “carrier,” the mole percent of leucine rises from 5.4 to 34.8 (544% increase) while in its absence these values change from 4.3 to 7.4 (727 o increase). The ability to assign an amino acid residue to a particular step is a function of the change in amino acid mole percent during consecutive steps. The inclusion of succinylated poly-ornithine has a profound effect on this value and concomitantly the ability to sequence lysozyme further. Comparable results were obtained using even smaller amounts of lysozyme (50 /~g, 3 nm) in the presence of “carrier.” Plots of amino acid yields indicate that these differences are due to a drastic decrease in the repetitive yield when “carrier” is omitted, presumably due to protein washout during the sequence cycle (Fig. 1). The repetitive yield in the presence of succinylated poly-ornithine calculated from the yields of glycine at step 4 and 16, and from the yields of leucine
290
SILVER
AND
HOOD
FIG. 1. Amino acid yields of lysozyme sequenator runs in the presence (0) and absence (0) of succinylated poly-ornithine. Background values were subtracted by comparison to a previous step. LORW due to manipulation were corrected against an internal standard of PTH-norleucine.
at steps 8 and 25 are 92 and 91%, respectively (Fig. 1). This compares favorably with sequence runs using macroamounts of protein. Omission of “carrier” reduces the repetitive yield to approximately 67%. Since several groups of proteins of significant interest (e.g., mouse H-2 alloantigens, nonhistone chromosomal proteins) require sodium dodecyl sulfate (SDS) for solubilization and subsequent purification (5,6), it was of interest to determine the feasibility of automated microsequence analysis in the presence of SDS. Light chain from homogeneous rabbit antibody of known sequence was used to determine the effect of succinylated poly-ornithine on the automated sequence analysis of protein in the presence of SDS. “Carrier” permitted the facile identification of the first six amino acid residues, while its omission made identification difficult past step 2 (Table 3). Weiner et al. (7) using a manual dansyl-Edman procedure were able to sequence microquantities of protein in the presence of SDS. Our results indicate that automated microprotein sequence analysis in the presence of SDS is also possible. The choice of succinylated poly-ornithine as putative “carrier” was based on several considerations. Ornithine, a homolog of lysine, is readily resolved from the other basic amino acids on an amino acid analyzer. Since it is not a naturally occurring amino acid its presence on amino
AUTOMATED
Automated
291
MICROSEQUENCING
Microsequence
TABLE 3 Analysis in the Presence of SDS0
A asP 1 SP
pro 2
val
leu
thr
3
4
5
glu 6
asp
Pro
val
leu
1
2
3
4
9.6
7.5
7.7
8.3
7.1
20.“ 1
10.5
9.s
8.4
!a
5.6
7.5
7.9
X.1
x.5
18.7
12.5
10.9
12.6
11.5
pro @Y
2.7 9.0
17.1 8.9
7.8 11.9
s .7 10.2
4.4 10.7
3 0 11.0
3.2 9.6
gg 11.2
5.5 11.3
7.4 14.6
ala
19.1 5.1
25.2 7.5
20.5 7.1
19.2 7.3
19.9 20.2
17.5 10.2
18.6 :3 .3
20.7 4.9
20.7 4.5
20.3 5.4
3.7 1.6
5.7 2.2
23.1 1.4
9,s 1.6
6.0 2.0
10.1 4.0
6.8 2.9
4.1
7.2
6.1
25.))
14.0
phe
2.0 1.8
3.4 3.0
2.4 2.7
2.6 2.5
2.7 2.. 5
2.:~ 2 I
1YS
‘2 3
‘2 7
1.7
1.6
1.9
5.7
8.1
6.8
7.4
6.4
ABA” val ileu leu t,yr
Total nmoles in sample
42.9
B
13.1
5.2 6 3
6.6 3.6
6.6 3 2
9 0
9 .2
9.7
3.7 s .!I
3 3 3 7
3 .!I 3 7
2 s 2.5
1.7
4,s
2.3
4.4
4.0
6.1
21 .!I
7.7
11.5
14.9
4 s
a Homogeneous rabbit light chain (100 rg) was dissolved in 200 Jiters of 0.5oj, SDS and sequenced in the presence of 5 mg of succinylated poly-ornithine (A) and in its absence (B). b PTH-threonine is degraded t#oa-amino butlyric acid (ABA) during the acid hydrolysis procedure. c Corrected against an internal standard of PTH-norleucine.
acid chromatograms is not confusing. The random polymer of ornithine was succinylated in order to render it highly anionic and soluble during
alkaline conditions (coupling with phenylisothiocyanate) and insoluble during the ethyl acetate and benzene washes during the sequenator cycles. These are ideal properties for any polypeptide undergoing the three cycle Edman reaction. The inclusion of “carrier” with the protein to be sequenced significantly limits protein washout and permits extended automated microsequence analysis. Recently, the development of a new reagent, “fluorescamine,” for amino acid detection permits a 50- to lOO-fold increase in sensitivity (8). This reagent used in conjunction with our synthetic “carrier” should permit. extended automated sequence analysis at the picomole level. Note: Preliminary results indicate that significantly less protein washout occurs when the N,N-Dimethylbenzylamine (DMBA) program is used (9) even when carrier is omitted.
292
SILVER
AND
HOOD
REFERENCES 1. EDMAN, P., AND BEGG, G. (1967) EUT. J. Biochem. 1, SO. 2. HOOD, L,, MCKEAN, D., FARNSWORTH, V., AND POTPER, M. (1973) Biochemistry 12, 741. 3. SMITHIES, O., GIBSON, D. M., FANNING, E. M., GOODFLIESH,R. M., GILMAN, J. G., AND BALLANTYNE, D. L. (1971) Biochemistry 10, 4912. 4. NEEDLEMAN, S. B. (Ed.) (1970) Protein Sequence Determination, p. 229, SpringerVerlag, New York. 5. WV, F., ELQIN, S., AND HOOD, L. (1973) Biochemistry 12, 2792. 6. SCHWARTZ, B. D., KATO, K., CULLEN, S. E., AND NATHENSON, S. G. (1973) Biochemistry 12, 2157. 7. WEINER, A. M., PLATT, T., AND WEBER, K. (1972) J. BioE. Chem. 246, 3242. 8. UDENFRIEND, S., STEIN, S., BOHLEN, P., DAIRMAN, W., LEIMGRIBER, W., AND WEIGELE, M. (1972) Science 178, 871. 9. HERMODSON, M. A., ERICSSON, L. H., TITANI, K., NEURATH, H., AND WALSH, K. A. ( 1972) Biochemistry 11, 4493,