Agric. Chem. Biotechnol.

46(3), 122-123 (2003)

Partial Purification and

Characterization of an AngiotensinConverting Enzyme Inhibitor from
Squid Ink
So-Youn Kim, Sun-Hye Kim and Kyung Bin Song*
Department of Food Science and Technology,
College of Agriculture, Chungnam National University,
Daejeon, 305-764, Korea
Received June 13, 2003; Accepted August 4, 2003

Key words : ACE inhibitor, isolation, squid ink.

Among biologically-active molecules, the angiotensinconverting enzyme (ACE) inhibitory peptides have been
extensively studied.1,2) ACE converts angiotensin I into
angiotensin II by cleaving the C-terminal dipeptide (His-Leu)
of angiotensin I and also inactivates bradykinin which
depresses blood pressure. Thus, the ACE inhibitor acts on the
inhibition of ACE, which results in a decrease in blood
pressure. It was screened from various food sources.1) Since
the discovery of an ACE inhibitory peptide in snake venom,
many ACE inhibitory peptides have been identified from the
enzymatic hydrolysates of various food sources. 3-6)
Squid ink is obtained as a waste product of squid product
processing. A peptidoglycan that is isolated from squid ink
has been reported to have anti-tumor activity7). Therefore, to
further elucidate other biological functions of squid ink and to
produce a high value-added product from the squid waste, its
anti-hypertensive property was examined and an ACE
inhibitor was isolated and purified from squid ink.
Fresh squid (Sepia esculenta) was purchased in Mookho,
Korea. Crude squid ink (50 mL) was filtered through a cheese
cloth and followed by centrifugation at 12,000 g for 30 min.
Squid ink was then filtered using a YM-3 (MW 3,000 cut-off)
membrane. The membrane-filtered solution was loaded onto
Sephadex G-15 (1.5 100 cm) that had been equilibrated
with a 10 mM phosphate buffer (pH 7.0). The eluate was
monitored by measuring the absorbance at 214 nm. Three
fractions were obtained from the column (Fig. 1). The ACE
inhibitory activity of each fraction was measured by the
method of Cushman and Cheung8) with modifications that
were established in our laboratory. 9-11) The reaction mixture
contained 150 l of 5 mM Hip-His-Leu as a substrate, 50 l
of rabbit lung ACE powder (5 munit) in a 50 mM sodium
borate buffer (pH 8.3), and 50 l of the sample solution. The
*Corresponding author
Phone: 82-42-821-6723; Fax: 82-42-825-2664
E-mail: kbsong@cnu.ac.kr

Short Communication

reaction was carried out at 37oC for 30 min, and terminated by

adding 250 l of 1 N HCl and 1 ml of ethylacetate. After
centrifugation, the absorbance of the supernatants was
measured at 228 nm. The IC50 value was defined as the
amount of the ACE inhibitor concentration that was needed to
inhibit 50% of the ACE activity.
The ACE assay result showed that the F1 fraction had the
highest inhibitory activity (32%). Therefore, the fraction was
pooled and loaded onto the normal phase HPLC (Thermo
Separation Products Inc., FL, USA) with an amino column
(4.6 250 mm, Capcell pak NH2, Shiseido Co., Tokyo,
Japan). Elution was performed on the A solvent (0.1%
trifluroacetic acid and 0.1% triethylamine in acetonirile: water
(97 : 3, v/v) and B solvent (0.1% trifluroacetic acid and 0.1%
triethylamine in acetonirile: water (30 : 70, v/v) conditions,
having a gradient of 0% of B to 55% at 0.5 ml min1. The F1
fraction was separated into three fractions by HPLC (Fig. 2).
Among them, the F12 fraction had the highest inhibitory
activity (36%). The F12 fraction was further purified using
FPLC with a Superdex peptide HR 10/30 (10 300 mm,
Amersham Pharmacia Co., Uppsala, Sweden) that was

Fig. 1. Elution profile of crude extracts of squid ink on a

GPC.

Fig. 2. Elution profile of fraction F1 on a normal phase

HPLC.

ACE inhibitor from Squid Ink

123

inhibitor from squid ink. Although the chemical nature of the

inhibitor should be further characterized, this small molecularweight inhibitor is quite promising in terms of manufacturing
a functional food product using squid ink. Further
characterization of the inhibitor, and the development of the
processing of a functional product, is currently being studied.

References

Fig. 3. Elution profile of fraction F12 on a FPLC.

Fig. 4. Mass spectrum of fraction F122 isolated from squid

ink.

equilibrated with 0.1% trifluroacetic acid (TFA) in

acetonitrile: water (30 : 70, v/v). There were two fractions
(Fig. 3). The F122 fraction had the highest inhibitory activity
(65%). Since this fraction was homogeneous, based on rechromatography, its molecular mass was determined using a
mass spectrometer (JMS HX-110A, Jeol, Tokyo, Japan). The
F122 fraction was identified as having a molecular mass of
294 (Fig. 4) and had 4.9 g ml 1 as the IC50 value. The ACE
inhibitor appears to be a peptide derivative since Edman
degradation was unsuccessful in determining the amino acid
sequence of the inhibitor. An additional NMR study is needed
to identify the inhibitor.
This is the first report regarding the isolation of an ACE

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