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S.

YANARI

685

hydrin-positive

material

could be detected by extraction

or by raphy

microdiffusion (10) showed

of ammonia (14); single ninhydrin-positive

(c) quantitative component

s

with ethyl acetate paper chromatog- in each prepara-

of the spot of L-Leu-L-Tyr being 3 times that of n-Leu- tion the intensity , L-Tyr;3 peptides and (d) determinations of for these di- groups were the dissociation constants5 free amino of indicated that theoretical amounts

present

and that each preparation

contained

a single component.

1

1

I

I

NINHYDRIN

NAPHTHOQUINONE-4- SULFONATE

2

OOW

00%

FIG. action agent.

and O, FIG. sulfonate

1. The mixture

MINUTES FIG. 1 MINUTES FIG. reaction of diastereoisomeric dipeptides with 2 ninhydrin.

contained

0.1 pmole

of dipeptide

and

1.0 ml.

of SnCls-ninhydrin

Each

volume

per

cent

methyl

42 per

cent methyl Cellosolve.

Cellosolve;

A total

and 0, volume,

1.2 ml., 25 volume

total

volume, 2.0 ml.,

re- re- A

2. The at 25”.

reaction Reaction

of diastereoisomeric

mixture:

1.0 Nmole

dipeptides of amino

with acid

1,2-naphthoquinone-4-

or

peptide,

200

pmoles

of NaHC03

(GH

1.6 ml.

At the

9.25),

end

of

8 pmoles

of

each

designated

3.0) and

0.2 ml.

tions were

read

of in

0.1 hf ascorbic a Klett-Summerson

acid

1,2-naphthoquinone-4-sulfonate;

reaction

period,

0.2

ml.

were added. photometer

After suitable (500 rnp filter).

total

of citrate dilution,

volume,

buffer

(pH

the

solu-

The reaction rates of L-Leu-L-Tyr

and D-Leu-L-Tyr

sulfonate rates are

(Fig. 2) obtained

are in with

distinct contrast to the naphthoquinone

the data reagent

with naphthoquinone in Fig. 1. Identical in aqueous solutions

at room temperature. peptide preparations

These data and further

indicate emphasize

molar equivalence the uniqueness

of the di- of the fact

that these diastereoisomers

do react at different

rates with

ninhydrin.

5 The bogen

dissociation (15). The

n-Leu-n-Tyr

(7.9)

were

determined

n-Leu-r-Tyr

(8.3)

constants pK2’ for

(unpublished

results).

by

a method

was

considerably

similar to higher

that than

of Ellen- that

of

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