Advances in the gas chromatographic analysis of amino acid phenyl- and methylthiohydantoins

ANALYTICAL

Advances

BIOCHEMISTRY

45, &-&

in the Gas Acid JOHN

C~romatagra~hic

Phenyl-

and

J. PISANO

Section of Physiological

(1972)

Analysis

of Amino

Methylthiohydantoins THOMAS

AND

Chemistry,

5. BRONZ~RT

Experimental

Therapeutics

Brarwh

AND

H. BRYAN Section on Peptide Lung Institute,

BREWER,

JR.

Chemistry, Moleculns Diseases Branch, National Institutes of Health, Bethesda, Received January

National

Heart

Maryland

2001.4

and

15, 1971

Previous studies in our laboratory (1,2) have shown that the phenylt,hiohydantoin (PTH) derivative of all the amino acids except arginine could be analyzed in submicrogram amounts by gas chromatography. The analysis was performed by direct injection of all derivatives except the aspartyl, glutanlyl, and cysteic acid ~o~npounds, which required prior conversion to the volatile trimethylsilyl derivative (1). Complete separation of all the amino acid PTH’s could not be achieved with a single stationa~ phase. Two phases, however, DC-560 and XE-60, were complementary, and allowed unequivocal identification of all the derivatives. A single column system composed of a blend of three phases (DC-560, XE-60, and OV-22), designated DXO, was developed, and came close to providing the desired resolution of all the phenylthiohydantoins. Only the resolution of the isoleucyl and leucyl derivatives and the silylated aspartyl and asparaginyl PTH’s were not. entirely satisfactory. This communication describes a superior blend of silicone phases as well as new r~rocedures that provide higher resolution, better peak symmet,ry and less baseline rise during temperature programming than hitherto obtained. The new procedure includes high temperature conditioning of the packing, the use of helium as carrier gas, and a sodium carbonate prewash of the support. Sequence analyses employing columns composed of the new blend are included. In addition, procedures are presented for the gas ~hromatographi~ analysis of the amino acid methylthiohydantoins fMTH’s), which may also be employed in sequence nnitlysis.

44

PISANO,

MATERIALS

BRONZERT,

AND

AND

BREWER

METHODS

Barber-Col,man model 5000 gas chromatographs used in this study were equipped with hydrogen flame detectors, double-column ovens with on-column injection ports, and temperature programmers. The injector temperature was 25&270”C,1 that of the detector, 300”. In all chromatograms, full-scale deflection was 3 X lo-lo A with a 5 mV recorder. Glass U or coiled columns, 4 ft x 2 mm i.d., used throughout were silanized with dichlorodimethylsilane (3). The carrier gas was helium. The support, Chromosorb W, 100-120 mesh, the stationary phases DC-560, SP-400, OV-210, and OV-225, and the silylating reagent, N,O-bis (trimethylsilyl) a&amide were purchased from Supelco, Inc., Bellefonte, Pennsylvania. Phenylthiohydantoin standards were purchased from Pierce Chemical Co. and Mann Research Laboratories, or synthesized in our laboratory according to published procedures. The methylthiohydantoins were synthesized with minor modifications by the method of Stepanov and Krivtsov (4). All reagents were analytical grade. Standard solutions (1 mg/ml) of the phenylthiohydantoins were made up separately in ethyl acetate with the following exceptions: the asparaginyl derivative (4 mg/ml) was dissolved in 2: 1 pyridine/ethyl acetate; the glutaminyl derivative (4 mg/ml) in 1:4 pyridine/ethyl acetate; the aspartyl and glutamyl derivatives (1 mg/ml) in 1:4 pyridine/ethyl acetate; and the histidyl PTH (4 mg/ml) in 1: 1 water/ methanol. Methanol alone can be used for these derivatives, but less concentrated (although more stable) solutions (0.5 mg/ml) are obtained. All solutions were stored in screw-cap vials at &4”. The stability of these solutions is currently under study.2 ‘At injector temperatures much below 250” (e.g., 200”), low responses are observed for the derivatives of asparagine, glutamine, tyrosine, lysine, histidine, and tryptophan. When the temperature is raised above 270”, asparagine, glutamine, lysine and histidine are the first to give lower responses. Most derivatives significantly decompose around 300”. ’ Standard solutions made with ethyl acetate are more stable than those containing pyridine, which last only overnight. In addition to these solubility and stability differences, the amino acid PTH derivatives are divided into three groups according to their gas chromat,ographic behavior. The Group I amino acid PTH’s (alanyl, valyl, prolpl, glycyl, isoleucyl, leucyl, methionyl, phenylalanyl) are most volatile and generally give symmetrical peaks. Members of Group II (asparaginyl, glutaminyl tyrosyl. histidyl. and trpptophanyl PTH’s) are least volatile. Histidyl, asparaginyl. and glutaminyl PTH show the greatest tendency to adsorb to the column packing, give t,ailing peaks. and low responses. Group III derivatives include those which must he silplated before analysis (aspartyl, glutamyl, and cysteic acid PHT’sJ and others which, when silylated, have significantly better chromatographic properties (S-carboxymethycysteine(SCMC), seryl. lysyl, threonyl PTH’s).

GC

OF

PHENYL-

AND

METHTLTHIOHYDAKTOINS

45

Support and Colufnn Reparation. With the exception of the NazCOa prewash, the procedure is essentially that employed by Horning et al. (3). Chromosorb W, 50 gm, is added to a 1 liter beaker and soaked overnight in approximately 500 ml of 0.5 M NaJX),,. This base wash generates a significant amount of fine particles, which are removed by decantation using distilled wat’er. The neutral support. is then soaked overnight in concentrated HCl. The fine particles are again decanted using distilled water. The support is dried at 140°C and 25 gm of the warm mat,erial is added to a 1 liter filter flask. To this flask is added 200 ml of 5% (v/v) dichloroclimetl~ylsilane in toluene and the mixture degassed under reduced pressure (provided by a suitably trapped aspirator). The support is swirled 2 or 3 times while under reduced pressure for 10-15 mm, the reagent decanted, and the support rinsed 3 times with toluene, taking care that the material is always wet with toluene, thus preventing any reaction with atmospheric moisture. The deactivation of the support is completed by suspending it in 300 ml of anhydrous methanol. After standing lo-15 min, the methanol is decanted and the silylated support rinsed with methanol until the solution is clear. The support is filtered on a sintered-glass funnel, rinsed with acetone, air dried, and completely dried in an oven at, 140”. Removal of the fine particles produced during the above procedures is essential for the preparation of efficient columns. After the base and acid washes, it is often necessary to decant 25 times with distilled water and, after silanieation, 3 times with methanol. It is important to note that Chromosorb W is a relatively fragile support and easily crushed. Crushed material contains newly exposed active sites and should not be allowed to mix with the bulk of the support. The support is coated by the filtration technique (3). In a typical experiment, 75 ml of solvent containing the stationary phase is added to 5 gm of support in a 125 ml filter flask. The mixture is degassed by gently swirling under reduced pressure (aspirator), filtered on a 150 ml sintered-glass funnel until dry, and transfcrrcd to a Petri dish and thoroughly dried at 140”. Column packings are usually described by the concentration of stationary phase dissolved in coating solvent; thus, a 10% DC-560 packing is prepared with 10 gm of DC-560 made up to 100 ml with acetone. The volume of solution used for coating by this technique is not critical but it should be sufficiently in excess to permit the transfer of the packing to the sintered-glass funnel. Acetone is the solvent for all of the silicone phases employed in this study. Columns are filled by adding the packing through a funnel connected to the column with rubber tubing. All columns are packed employing house vacuum, but, may also be filled by gravity flow if so desired. Care

46

PISANO,

BRO~ZERT,

AND

BREWER

should be taken not to crush any support during the packing procedure. As the columns are filling, they are gently tapped with a pencil or similar object in order to pack evenly but not too tightly, as this will impede gas flow. A typical column conditioning program for a 4 ft X 2 mm i.d. column is: initial helium flow 150 cc/min, initial temperature 5O”C, maint,ained for 30 min, and then raised to 300” at the rate of 05”/min. The column is held at 300” for 16 hr or until a suitably low baseline rise (2%) is obtained in the temperature-programmed analysis. Phenylisothiocyanate degradations were performed by the three-stage procedure of Edman (5)) employing the 1 N HCl for the conversion step (6). Subtractive analyses during Edman degradations were based on assays of aliquots of peptidc taken at the beginning of the next step (after addition of coupling buffer}. These aliquots were freeze-dried and hydrolyzed in 5.7 N HCl for 24 hr. Amino acid analyses were performed on the Beckman-Spinco automatic amino acid analyzer, model 12OB, adapted for high sensitivity and rapid elution schedule (7). Synthetic bradykinin was obtained from Schwarz BioResearch. Homogeneous bovine thyrocalcitonin was prepared by the method previously reported (8,9). RESULTS

Previous studies have shown that acid washing of the support prior to coating removes many interfering contaminants; washing in strong base, on the other hand, can destroy the support (3). We have found that a wash with the weaker base, 0.5 M sodium carbonate, prior to acid washing, improves the performance of the support. In a typical comparison with a 4 ft DC-560 column, the support prewashed with sodium carbonate gave 1360 theoretical plates while that washed with only acid gave 1024 plates, calcuIat.ed using the alanyl PTH peak. Carrier Gas. A comparison of the three common carrier gases (argon, nitrogen, and helium) made by plotting height equivalent to a theoretical plate (HETP) against flow rate revealed that helium gives the most efficient columns under the conditions employed (Fig. 1). In addition, the use of helium allows the widest range of flow rates without significantly increasing the HETP. Coleus Perfo~~a~ce.3 In addition to the sodium carbonate prewash, helium carrier gas and high-temperature conditioning (i.e., 300°C for a Many columns do not perform optimally on the first and, sometimes, second day of use as evidenced by slight peak tailing. This tailing virtually disappears with good columns, but persists in poor columns, due usually to improper preparation of the support.

GC

0.001

OF

PHENYL-

I

25

0

AND

47

METHYLTHIOHYDANTOIKS

I

I

I

I

50

75

100

150

GAS

FLOW

(ml

I

I

200

/min)

FIG. 1. Height of column equivalent to a theoretical plate (HETP) vs. carrier gas flow rate (measured at the column outlet) determined on a CFC column, 4 ft X 2 mm i.d., at 17O”C, using alanyl, valyl, or prolyl phenylthiohydantoin. Amino Acid Ser Thr TM%- PTHs scMy pso3H I I \I I II Ala Gly Vol Ile Leu Amino Acid Ala Gly Val PTHs , ,, II SCMC Sex

Pro Leu , , I Ile

Met Phe Asn Gln Tyr His r

GIII

Column

1

W

His

Tqr

I------170”

I Trp

LYS

Phenylthiohydontoins 10% DC-560

Asp Asn Glu Gln Lys 1 I I I Tyr His Met Phe

I

TvI

5”/ m,n -----I-z70°4

FIG. 2. Separation of amino acid phenylthiohydantoins on a 4 ft X 2 mm i.d. 10% DC-560 column. Sample sizes: 1 pg each alanyl, glycyl, valyl, prolyl, leucyl, methionyl, phenylalanyl, and tyrosyl PTH’s; 2 pg tryptophanyl, asparaginyl, glutaminyl, and histidyl PTH’s. Helium flow 65 ml/mm.

48

PISANO,

BRONZERT,

AND

BREWER

16 hr), the DC-560 column has been reduced from 6 ft X 4 mm i.d. to 4 ft ,x 2 mm id. Figure 2 shows a chromatogram obtained with the phenylthiohydantoins and their silyl derivatives obtained with a DC-560 column prepared and operated under the new conditions. Compared to the former procedure (l), resolution is better (610 plates/ft for a 4 ft X 2 mm column vs. 405 plates/ft for a 6 ft X 4 mm column), analysis time is shortened 10 min, and the baseline rise is lower during programmed temperature rise (270 vs. 7% of full scale). Similar improvements were also observed with the DXO blend. Without changing column dimensions, an increase from 1500 to 2190 theoretical plates was achieved with the new techniques. The most noteworthy improvement was the better separation of the isoleucyl and leucyl derivatives. However, the thermal lability of the silicone stationary phase XE-60 and the noticeable peak tailing of the Group II derivatives are remaining limitations of this blend. New Blend. The new blend (designated CFC) consists of equal volumes of 7.33% SP-400,5.33% OV-210, and 0.66% OV-225. Columns made with this blend produce less bleed than the DXO blend and better resolution for: (1) the PTH’s of Group II, (2) t,he isoleucyl and leucyl

AminF;>dySCMC

Ser Thr

Asp

II II TMSi-PTH~~nVolGIyIleLeu , Amino ’ PTHs

Acid

Ala Vol Pro Gly

Glu Lys

III

III

Asn Met Phe Gln Tyr His Ile Leu

i

I

Tv

Met Phe Asn GlnTyr His I LYS

Trp

Phenylthiohydontoins

I

165”

I+--

7,5’/min

-----l-

270’--+

FIG. 3. Separation of amino acid phenylthiohydantoins on a 4 ft X 2 mm i.d. CFC column, Sample sizes: 1 pg each alanyl, valyl, prolyl, glycyl, isoleucyl, leycyl, and tyrosyl PTH’s; 2 pg each asparaginyl, glutaminyl, histidyl, and trytophanyl PTH’s; 0.5 pg each methionyl and phenylalanyl PTH’s. Helium flow 105 ml/min.

GC

OF

PHENYL-

AND

49

METHYLTHIOHYDANTOINS

derivatives, and (8) the silyl aspartyl and silyl asparaginyl compounds. Only the tyrosyl and lysyl PTH’s are poorly separated, but their silyl derivatives are completely resolved (Fig. 3). The behavior of the prolyl derivatives on this (and other columns) is unique

in that

its position,

relative

to the other

derivatives,

is

temperature dependent. Thus, the initial isothermal temperature can be either raised to move prolyl PTH closer to glycyl PTH or lowered to move it closer to valyl PTH. Subsequent adjustment of the helium flow rate will then position the Group I peaks to any desired time without affecting column efficiency. The positions of the silylated thiohydantoins are also shown in Fig. 3.

Figures 4 and 5 show the application

of trimethylsilylation

specifically

Glu TM%-PTI i

CFC Column

CySO,H TMSi-PTH Asp TM%P TH I

L ‘5

'IO

‘15

II '20

.L' 25

FIG. 4. Separation on CFC column of trimethylsilyl products of z pg glutamyl, and lysyl PTH’s and 4 pg cysteic acid PTH. The dashed peak lysyl PTH before silylation. Helium flow 105 ml/min.

aspartyl, is 4 pg

50

PISANO,

BRONZERT,

AND

BREWER

CFC Column

Ser TMSi-PTH Thr TMSi-PTH

165"

i FIQ. peaks)

5. As for and after

Fig. 4: l-2 silylation.

mg

SCMC,

i

seryl,

and

threonyl

PTH’s

before

(dashed

to the amino acid derivatives which must be silylated (glutamyl, aspartyl, cysteic acid) and to those which give significantly improved responses (seryl, threonyl, SCMC, lysyl PTH’s) when silylated. Trimethylsilylations were carried out at 50°C for 15 min in a 50:50 mixture of ethyl acetate and N,O-bis (trimethylsilyl) acetamide in sealed tubes (1). Application

of the CFC

Column

Bradykinin. A comparison of the results obtained from the gas chromatographic procedure with the commonly employed subtractive method of analysis was performed with 1.2 pmole synthetic nanopeptide, bradykinin. The PTH amino acid cleaved at each step (except arginine) was quantitated from the peak areas. Steps 2 to 8 are shown in Figs. 6 and 7. The chromatograms are virtually free of extraneous peaks and the cleaved amino acid is outstanding at each step of the sequence. Step 6 is noteworthy. At first sight, the chromatogram 6a would indicate either a seryl or a phenylalanyl residue since the peak areas are similar. However, the data do in fact, indicate serine when one considers that (I) the yield of the serine derivative is normally about 2040% in the Edman degradation4 and (2) the compound per se normally gives a peak area only 50% that of an equal weight of the phenylalanyl or most other PTH’s. Supporting evidence for step 6 serine is seen in ‘The yield cantly improved

of serine during Edman degradation by the use of the reducing agent

has been dithioerythritol

shown

to (10).

be

signifi-

GC

OF

PHENYL-

AND

METHYLTHIOHYDANTOIKS

Arq-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arq

STEP 5-PTH

Phe

STEP 4 -PTH Gly

5

IO

15

20

25

30

3 MI

3

5

IO

4

15

20

25

30

315

15

;-yL, 20 25

xl

I 35

TES

Arg-Pro-Pro-Gly-Fhe-Ser-Pro-Phe-Arg 123456789

I /I

STEP 8-PTH Phe

L, 5

IO

I5

20

25

30

35

0

5

Pro $\ IO

MIWTES

FIGS. 6 AND 7. Analysis of Edman degradation products of bradykinin with exception of arginine PTH, which cannot be determined by gas chromatography. Step 4 contains slight prolyl overlap. Steps 6a and 6b illustrate use of trimethylconditions same as those for silylation in identifying serine. Chromatography Fig. 3.

52

PISANO,

BRONZERT,

AND

BREWER

chromatogram 6b, which shows the expected greater area for the silylated serine derivative. Silylation of serine gives the N,O-bis (trimethylsilyl) compound (11)) whereas phenylalanyl PTH forms the N-trimethyl compound. Furthermore, silylated serine is more stable than the parent compound during chromatography. It is unclear if the phenylalanine present at step 6 is due to overlap from st,ep 5, or represents a small amount of phenylalanine present at position 6 in the synthetic peptide, since the other steps in the sequence are nearly free of overlap. A comparison of the yield of the amino acid at each step in the sequence obtained by gas chromatography of the cleaved PTH and the results obtained from amino acid analysis of the residual peptide following the degradation are shown in Table 1. There is a relatively good agreement between the amount of amino acid cleaved from the peptide and the PTH quantitated by gas chromatography. Thus, the gas chromatographic procedure can be readily used to quantitatively determine the amino acid(s) present at each step in the sequence of a peptide or mixture of peptides. Bovine Thyrocalcitonin. The complete covalent structure of bovine thyrocalcitonin was obtained utilizing the gas chromatographic technique for identification of Edman derivatives (8). Representative chromatograms obtained from the Edman degradation of reduced and alkylated thyrocalcitonin, and the trypsin peptide T,, are shown in Fig. 8. As in the previous degradations of bradykinin, the chromatograms are nearly

Edman

Degradation

TABLE 1 of 1.25 pmoles Amino

Arg (1)

1.10

Amino acid

0

Arg

2.1

Pro

3.2

Pro

Pro

(2)

(3)

pmoles 0.70

amino 0.75

-

0.80

1

2

",??..9" 1.0

0.8

GUY (4)

acid sequence Phe Ser

0.8 “1.1”

(5)

analysis 4 1.2 1.2

Phe (8)

degradation 0.41 0.67 of PTH 0.31

0.46

(residue/mole) 5 6

7

8

GUY

1.0

Phe

2.0

2.1

2.1

2.1

2.2

1.4 0.3 “1 ,5”

Ser

0.9

1.0

1.0

0.9

0.9

1.1

“0.2”

Pro (7)

analysis 0.21

0.48

“1 .O” 2.9 1.0

1.0

(6)

acid cleaved by Edman 0.57 0.40 0.44

Gas chromatographic 0.64 0.67 Subtractive 3

Bradykinin

0.9

0.9

0.9

1.0

1.1 0.2 1.3 “0.4”

"0.5"

0.3

Arg (9)

-

-

0.2

0.1

-

1.1 0.2

,(,,4” 0.2

-

-

GC

OF

PHENYL-

AND

~E~~YL~~IOHYD.~~~I~S

53

Reduced and Alkyialed Thy~alcito~n Step II

\ /

Reduced and s’ep 22

___--.. ..A Tryptic Peplide Ts step 9 TC Resiiue 30

ThiSi -PTH Giuta~ deid

FIG. 8. Selected steps from Edman degradation of bovine thyrocalcitonin. Either the CFC (Fig. 3) or DC-560 (Fig. 2) column was employed. Aspartyl, g~~Itarn~~1, and seryi residues wirre identified after silylation. Ristidyl PTH in step 20 was recovered from the water layer in the conversion step of the Edman procedure.

54

PISANO, Amino Acid

BRONZERT,

Ala Gly Vol II I

‘TMSi-MTHs

Ile Leu II

SCMC

E/y

Ala Gly Val I, I

MTHs

SCMC

Pro I

Ile Leu I I

AND

BREWER

Gin Tyr Met Phe Asn His Lys I,, I, I I Asp Glu Met Phe Asn Gln Tyr Lys I II I II

Thr

Trp 1

Tv I

His

Skr

15%

I

DC- 560

Column

l20”------(

7.5”/min-(

-230O-1

FIG. 9. Separation of amino acid .methylthiohydantoins on a 15% DC-560 column. Sample sizes: 1 pg each, except for 0.5 gg methionyl and phenylalanyl MTH’s. Helium flow 60 ml/min.

free of overlap and extraneous peaks allowing for unequivocal identification of the amino acid at each step in the sequence. In addition, the presence of a single amino acid at step 1 indicates the homogeneity of the isolated peptide. The aspartic acid (residue 15) and glutamic acid (residue 30) were identified following trimethylsilylation. Additional peaks are frequently seen after silylation. They appear just after the solvent front (Fig. 8, residues 15, 23, and 30) but they do not coelute with any of the silylated PTH’s and have not been a problem in the identification of the cleaved amino acid. The shoulder on the serine peak in the chromatogram of residue 23 (Fig. 8) is due to incomplete silylation of the sample. The small peak following phenylalanine (Fig. 8, residue 22) has occasionally been noted in standards, and probably represents a degradation product of this amino acid. Chromatography

of the MTH’s

Several laboratories have recently considered substituting the more volatile methylisothiocyanate.,for phenylisothiocyanate ip the .Edman degradation of peptides and proteins (4,12-16). Although the advantages

CX

OF

PHENYL-

AND

55

METHYLTHIOHYDAKTOINS

for such a substitution are not clear at this time, it was nonetheless of interest to observe chromatographic properties of the resulting amino acid ,methylthiohydantoins. The behavior of the MTH’s and their trimethylsilyl derivatives on a 15% DC-560 column is shown in Fig. 9. Unlike the phenyl counterparts, the isoleucyl and leucyl methyl compounds are almost completely resolved, while the tyrosyl, histidyl, and lysyl derivatives are separated only after trimethylsilylation. Also, unlike the PTH’s, silylation of the methyl compounds produces later, rather than earlier, eluting peaks. The DC-560 column also gives separate peaks for the asparaginyl and glutaminyl MTH’s and their silyl derivatives. The greater volatility of the met.hyl derivatives is also apparent. The phenyl compounds would require temperatures approximat,ely 50% higher to elute at the same time. Even better separation of the MTH’s is possible with a 2% OV-225 column (Fig. 10). All of t’he methyl derivatives are separated and they elute as symmetrical peaks. This more polar stationary phase is highly suited from the h/ITH’s and compares very favorably with previous reports (12,1&16).

Amino Ac,d Pi'tO! I ’ MTHs SCMC

Gly I!'? LeU I Thr

Met Phe

Lys His Asn Gln Tyr

TIP

Amino Acid Melhylthiohydantoins 2% OV- 225 Column

+-170”---1

FIG. 10. Separation of amino acid Sample sizes: 1 fig ea,ch. Helium flow must be lowered in order to identify amino ataid MTH’s.

S”/min

methylthiohydantoins 60 ml/min. The the trimethylsilyl

t--27oq on 2% OV-225 column. initial isothermal temperature derivatives of the earliest

56

PISANO,

BRONZERT,

AND

BREWER

Although the chromatographic properties of the MTH’s are generally comparable to the PTH’s, the seryl compound appears to be troublesome as standards are unstable and tend to polymerize. Others have previously reported this observation (14)) but state that the residue obtained in a degradation is identifiable. DISCUSSION

Successful application of the gas chromatographic procedure to the analysis of the PTH’s requires an appreciation of some basic facts: (1) Metal columns cannot be used as they cause destruction of the amino acid PTH’s. (2) Acid washing and silylation of the support markedly reduce the adsorptive sites, which would otherwise cause excessive tailing of the derivatives. (3) A sodium carbonate prewash of the support produces even less tailing and a significant increase in theoretical plates. (4) Assiduous removal of the fine particles when preparing the chromosorb increases column efficiency. (5) Of the three common carrier gases tested, helium is superior to nitrogen and argon for resolving the amino acid PTH’s. The physical packing of the glass columns is not critical as long as none of the support is either crushed or packed too tightly. With a 4 ft X 2 ‘mm column at temperatures below lOO”C, a helium flow of 150 ml/min is normally attainable with an inlet pressure below 40 psi. If this flow rate cannot be achieved the column must be repacked. The use of a silylated glass wool plug is required at the column outlet to prevent loss of the support. We do not use a glass wool plug at the inlet, however, since it tends to accumulate nonvolatile deposits harmful to the derivatives. Furthermore, the removal of the first 1 or 2 in. of support after numerous injections of badly contaminated samples is more difficult when glass wool is used .5 This latter technique is recommended when a column appears to be losing efficiency or causes excessive peak tailing (most notable with the amides, which may completely disappear). In most cases, however, such columns are discarded. Columns ‘In principle, one should have a minimal dead space above the column packing. In the present technique, with the 2 mm i.d. columns and high helium flow rates, the dead space can vary several inches without significantly affecting efficiency. On t,he other hand, one should be careful not to have packing in the uppermost part of the column, which is in the injector block. Excessive temperatures will strip this section of the packing, exposing the bare support, which could destroy many of t.he thiohydantoins. Another hazard of an overfilled column is the mechanical destruction of 6he packing and exposure of unsilylated sites caused by the needle of the injection syringe. The support is mechanically fragile and careless handling should be avoided. Damaged support should never be allowed to mix with good support.

GC

OF

PHENYL-

AND

METHYLTHIOHYDANTOIKS

57

not abused have been used continuously for several months and stored indefinitely. Column life will bc del)endent also upon the purity of sequence samples. A high percentage of nonvolat’ilc impurities tends to decrease column longevity. The gas chromatographic analysis of the amino acid PTH’s has been significantly improved by the new CFC column prepared with the sodium carbonate washed Chromosorb, conditioned at 300”, and using helium as carrier gas. A single analysis may now be completed in 40 min from the time of injection. This blend of silicone stationary phases gives superior resolution of all the PTH’s, incorporating the best properties of the DC-560 and OV-225 single phases. Specifically, the CFC blend gives good resolution of the Group I amino acids, as does. DC-560, as well as the isoleucyl-leucyl separation and the superior Group II resolution characteristic of OV-225. The temperature program shown in Fig. 3 was selected because it allows sufficient separation of the solvent front and silylat.ion byproducts from the first elut’ing compound, silylated alanyl PTH. The space between leucyl and methionyl PTH is necessary for identifying the trimethylsilyl derivatives of the aspartyl and glutamyl residues and for calculating the percentage of deamination of asparagine. This program is suitable for all derivatives before and after t,rimethylsilylation. Silylation of all samples before injection is a possibility not yet fully explored by us or other laboratories (1,11,17). The potential advantage would be the saving of time in that all the PTH’s could be identified with a single injection. Thus, reinjection would not be necessary for the identification of aspartyl, glutamyl, and cysteic acid PTH’s. Furthermore, the improved response of silylated SCMC, lysyl, seryl, and threonyl PTH’s would also be realized. However, identification of the majority of derivatives is not facilitated by silylat.ion. Although individually they can all react quantitatively and the products have excellent chromatographic properties, the rate of reaction and the stability of the derivatives are somewhat variable (1). This is particularly t’rue with the asparaginyl and glutaminyl compounds. Occasionally, crystalline standards and more often the amides obtained from a protein degradation give no peaks upon silylation, whereas the unsilylated derivatives respond normally. Silylation of samples obtained from a degradation also results in the appearance of many peaks not previously present. Fortunately, none has yet. been observed with retention times the same as any of the amino acid phenylthiohydantoins. The CFC blend does not separate the amino acid MTH’s. For these derivatives the simpler single-phase columns DC-560 and/or OV-225 are satisfactory. The DC-560 phase (increased t,o 15%) is again excellent

58

PISANO,

BRONZERT,

AND

BREWER

for the Group I derivatives, but a 2% OV-225 column is superior, as it will adequately resolve all of the amino’ acid MTH’s. The development of the automated degradation of polypeptides has stimulated interest in automated analysis of the amino acid PTH’s. The automatic sample injector offered by Barber-Colman, now NuclearChicago, analogous to published designs (l&19), has given encouraging results with standards. Approximately 0.5 pg of sample in 3-7 ~1 of solvent may be identified every 50 min. At this rate, is seems possible that all samples may be analyzed directly and after trimethylsilylation and still keep pace with the protein sequenator. Application to PTH’s obtained from a degradation are presently in progress. Stability of the derivative is the main concern as it is well known that the phenylthiohydantoin samples obtained from a degradation could contain impurities which cause their destruction during the 1-16 hr wait prior to injection. Nevertheless, the potential of an automated analysis for the amino acid phenylthiohydantoins appears promising. SUMMARY

Advances in the gas chromatography procedure for the identification of amino acid thiohydantoins include: (1) a new blend of silicone stationary phases (CFC), which provides superior resolving power than hitherto obtained with single phases or earlier blends; (2) use of a carrier gas helium, which is a superior carrier gas to the more commonly used nitrogen and argon in that it gives the best resolution and the greatest range of flow rates with no sacrifice in efficiency; and (3) high-temperature conditioning, which gives more efficient columns with significantly less bleed (baseline rise) during temperature programming. Application of the technique is demonstrated with representative analyses of phenylthiohydantoins (PTH’s) obtained in the manual Edman degra,dation of bovine thyrocalcitonin. The methylthiohydantoins (MTH’s) were also examined. All derivatives, except the seryl (unstable) and arginyl (nonvolatile) were separable on a column using the stationary phase, OV-225. REFERENCES 1. PISANO, J. J., AND BRONZERT, T. J., .I. Biol. Chem. 224, 5597 (1969). 2. FISANO, J. J., AND BRONZERT, T. J.. Fed. Proc. 29, 916 (19701, Abstracts. 3. HORNING, E. C., VANDENHEUVEL, W. J. A., AND CREECH, B. G., Methods Biothem. Anal. 11, 69 (1963). 4. STEPANOV, V. M., AND KRIVTSOV, V. F., J. Gen. Chem. (USSR) 3!j, 53$X,988 (1965). 5. BLOMB~~CK, B., BLOMBXCK, M., EDMAN, P., AND HESSEL, B., Biochim. Biophys. Acta 115, 371 (1966). 6. ILSE, D., AND EDMAN, P., Aust. J. Chem. 16, 441 (1963). 7. HUBBARD, R., AND KREMEN, D. M., Anal. Biochem. 12, 593 (1965).

GC

11. 12. 13. 14. 15. 16. 17. 18. 19.

PHENYL-

AND

METHYLTHIOHYDANTOINS

59

H. B., JR., AND RONAN, R., Proc. Nut. Acad. Sci. U. S. 63, 940 (1969). H. B., JR., SCHLUETER, R., AND ALDRED, J. P., J. Biol. Chem., in press. HERMODSON, M. A., ERICSSON, L. H., AND WALSH, Ii. A., Fed. Proc. 29, 728 (1970)) Abstracts. HARMAN, R. E., PATTERSON, J. L., AND VANDENHEUVEL, W. J. A., Anal. Biochem. 25, 452 (1968). DIJKSTRA, A., BILLIET, H. A., VAN DONINCK, A. H., v.4~ VELTHUYZEN, H., MATT, L., AND BEYERMAN, H. C., Rec. Tram. Chim. 86, 65 (1967). RICHARDS, F. F., BARNES, W. T., LOVINS, R. E., SALOMONE, R., AND WATERFIELD, M. D., Nature 221, 1241 (1969). WATERFIELD, M., AND HABER, E., Biochemistry 9, 832 (1970). ATTRILL, J. E., BUTTS, W. C., R~INEY, W. T., JR., AND HOLLEMAN! J. W., Anal. Lett. 3, 59 (1970). VANCE, D. E., AND FEINGOLD, D. S., Anal. Biochem. 36, 30 (1970). GUERIN. M. R., AND SHULTS, W. D., J. Chromatogr. hi. 7, 701 (1969). TINTI. P., J. Gas Chromatogr. 4, 140 (1966). RUCHELMAN, M. W., J. Gas Chromatogr. 4, 265 (1966).

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