Hydrolysis of phenylthiohydantoins of amino acids

Vol. 14, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HYDBOLYSISOF PHENYLTKCOHYDANDINS OF AMINOACIDS H. 0. Van Orden and Frederick H. Carpenter Departmnt of Biochemistry,

Received

December

4,

University

of California,

Berkeley,

California

1963

The Edman(1950) pmposd for sequential detemination acids in a peptide chain has been applied in a direct

of the amino

or subtractive

manner.

In the direct manner, the nature of the N-terminal amino acid is determined by identification

of the liberated

phenylthiohydantoins

amino acids by paper or column chromatography (Sjaquist, and SjSquist,

1956).

acid is identified

In the subtractive

by the difference

peptide before and after

application

et aI--, 1951; Hirs, et al., liberated

1960).

phenylthiohydsntoin

of the N-terminal 1955, 1960; Edman

pmcedure, the N-terminal amino

in amino acid camposition of the of the degradation reaction In a edified

direct

procedure, the

is subjected to various hydrolytic

ments to release the constituent

amino acid which is identified

chrcmatography (Edman, 1950; Ingram, V. M., 1953; Levy, 1954). report explores the use of the amino acid analyzer to identify titate

the amino acids released upon hydxdysis

(Fox,

treatby paper The present and quan-

of 3-phenyl-2-thiohydan-

toins of various amino acids.

The thiohydantoins

to several types of hydrolytic

treatment in an attempt to determine opt+-

mumconditions for cleavage to the constituent conditions included an alkaline

hydrolysis

amino acids.

399

The hydrolytic

similar to that recently

posed by Stark and Smyth (1963) for the hfirolysis amino acids.

have been subjected

pro-

of the hydsntoins of

Vol. 14, No. 5, 1964

EXPERIMENTAL

The 3-phenyl-2-thiohydantoins

of the amino acids were prepared in

this laboratory

and characterized

by melting point,

snd ultraviolet

absorption spectra.

elementary amlySeS

Amino acid analyses were detezmined

by the method of Moore, -et al. (1958), and Spa&man, et al. (1958), using a Beckman/Spinco Amino Acid Analyzer. Solutions of the thiohydantoins lunoles of compoundperml. into a hydrolysis

were prepared to contain 1 to 1.5

A l-ml aliquot

tube (micro-Carius),

a stresm of nitlrogen to yield

and the solvent was removed under

the solid phenylthiohydantoin

subjected to acid or basic hydrolysis. (I.) three solutions in ethyl acetate, toins of aspartic

leucine,

(in acetone containing

thiohydantoins

each containing

tyrosine,

(3)

the phenyltbiohydan-

glycine,

alanine, valine,

and phenylalanine;

l$ of water),

of arginine hydrochloride,

8phenylthiocarbsmyl~lysine;

which was

The solutions prepared were:

acid, glutsmic acid, proline,

methionine, isoleucine, solutions

of each solution was introduced

each containing the phenyl-

histidine

two solutions

(2) two

-chloride,

(in ethyl

,5$ acetone) of each of the single phenylthiohydantoins

and

acetate with

of serine and

threonine. Hydirolyses were performed by adding either 2 ml of 6 g HCl (metal-free) thiohydantoin.

to each tube containing

NaOHor

the solid phenyl-

The contents were frozen and the tubes were evacuated

on an oil pump and sealed. or 150' for 24 or 48 hours. the case of the alkaline Mz.ary evaporator. of water.

3 ml of 0.12

The tubes were heated in an oil bath at 120 The hyarolysates

hydrolysis)

(acidified

with

were concentrated to dryness on a

The evaporation was repeated twice after

The solid residue, (except for

solved in 5 ml. of the pH 2.2 citrate mixture was centrifuged

6 g HCI in

precipitated

buffer

silica)

(Moore and Stein,

the sddition was dislg%),

(when necessary to remove suspended silica),

the supernatants were stored at -loo until

the and

subjected to amino acid analyses.

BIOCHEMICAL

Vol. 14, No. 5, 1964

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The results shown in Table I are aversge values obtained from the two or three completely separate defendnations.

Values agreed within

-L 3% of

the mean. TABLE1 HYDHJLYSISOF PH3NYLTHIOHYDAN%UNS

IIistidine

70

Arginine as Omithine Aspartic

<2

53

acid

89

48

Threonine as Glycine Serine


Glutsmic acid

66

39

81

Pxvline

96

64

88

Glycine

lo4

Alanine

86

40

ValiXE

98

12

Methionine Isoleucine as Alloisoleucine

78

I

1 I I

I

88 I I 67

I

43 58

72

80 99 66 83 52

6"

Leucine 76 86

I

66 78

Vol. 14, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESUTJTS

The recovery of amino acids upon hydrolysis toins is shown in Table I. partially

hydrolyzed

of their

phenylthiohydan-

In general, the phenylthiohydantoins

in 6 2 HCl at 120' for 24 hours--conditions

give complete hydrolysis

of most proteins.

were only which

Upon increasing the tempera-

ture to 15C0, the yields of amino acids from the hydrolysis

in 6 g HCl

were much improved and, in somecases, approached 904 of the theoretical Extending the reaction time to 48 hours at 150' resulted in lower

value.

values than those obtained in 24 hours.

Both serine and threonine were

completely destroyed in the acid hydrolysis. phenylthiohydantoin

of glutsmic acid, all of the other

toins studied gave a larger yield the alkalLne hydrolytic

conditions

under any of the acidic conditions. but threonine was largely covered in 53 yield

With the exception of the

of the constituent

amino acids under

(0.1 g NaOHfor 24 hours at X20') than A&n

serine was completely destroyed,

recovered as glycine

86 ornithine.

phenylthiohydan-

(6%).

Arginine was re-

In both the acidic and basic hydroly-

sis, isoleucine was recovered in part as slloisoleucine.

The total

isoleucine plus

amount in the

basic hydrolysis.

soleucine approached the theoretical

of

In general, our results are consistent with the earlier

studies (Edman, 1950; Ingram, 1953; Levy, 1954) which used paper chromatographic techniques to detect and quantitate are in close sgreement tith hydrolysis

and

the results of Stark and Snyth (1963) on the

of the hydantoins of amino acids.

above, the recoveries of the constituent drolysis

the amino acid recoveries,

of the 3-phenyl-2-thiohydantoins

With the exceptions noted

amino acids in the alkaline

hy-

of the amino acids were sufficiently

high that the values could be used without correction

in the determination

of an N-terminal amino acid.

This work was supported in part by a grant (A-608) from the National Institutes

of Health and a grant (G-19561) from the National Science 402

Vol. 14, No. 5, 1964

Foundation.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COhVWNlCATlONS

The authors wish to thank Mrs. Bertha Martin

for aid with

the amino acid analyses.

REFZRENC.ES

Eanlan, P., Acts Chem. Scand., 4, 283 (1950). Edman, P.,

and Sj8quist,

zbx, s. w., Hurst, 3573 (1951). Hirs,

C. (1960)

Ingram,

W.,

I-I.

J.,

Acta Chem. Scancl., l0,

1. L., and Itschner,

Moore,

S.,

and

K. F., J. Am. Chem. tic., W. H., J. Biol.

Stein,

73,

Chem., 235, 633

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V. M., J. Chem. Sm.,

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1953, 3717.

Biophys. D. Ii.,

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W. i-I., J. Biol.

W. H., Anal. Chem., 2,

Chem., 2ll,

J.,

Biochim.

Biophys.

Acta, l6,

283 (1955).

Sjdquist,

J., Biochim.

Biophys.

Acta, bl,

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Spa&man, D. H., Stein, (1958) .

W. H., and

Moore,

G. R., and Sayth, D. G., J. Biol.

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1185

893 (19%).

Sjbquist,

Stark,

1507 (1956).

S., And.

Chem., 30, llgCl

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