2018 Springer Science+Business Media, LLC
Chemistry of Natural Compounds, Vol. 54, No. 3, May, 2018
ISOLATION AND PURIFICATION OF ACTIVE PROTEASES
FROM SHEEP ABOMASUM AND THEIR BIOLOGICAL ACTIVITY
O. S. Charyshnikova,
Sh. Ya. Mirzaakhmedov,
and H. A. Aisa
Sheep abomasum active proteases (SAPs) were purified by chromatography over a column of Sephadex
G-25 and by ultrafiltration. The SAPs were found to contain proteases of molecular mass 14 kDa and active
peptides with molecular masses varying in the range 651.39–4248.08 Da. Peptide fractions of SAPs reduced
significantly (up to 72.0
2.3% inhibition) accumulation of malondialdehyde (MDA) in brain homogenate
of laboratory rats.
Keywords: sheep abomasum (SA), peptide extraction and isolation, electrophoresis, liquid-chromatography–mass-
spectrometry analysis, sheep abomasum active proteases (SAPs), proteolytic activity, antioxidant activity, lipid peroxidation
(LPO), malondialdehyde (MDA).
The chemical compositions and biological activities of sheep abomasum active proteases (SAPs) remain little studied
despite reports in the scientific literature that active proteases isolated from sheep abomasum (SA) are used to treat acute and
chronic gastritis [1, 2]. A detailed investigation of bioactive proteases and the peptide composition of SA provided a basis for
designing new promising drugs for GIT diseases. The goals of the present work were to isolate SAPs, to identify peptide
molecular masses using LC-MS, and to study their proteolytic and antioxidant activities.
A simple and convenient two-step method for isolating and purifying SAPs was developed. The first step used
gel-filtration chromatography and ultrafiltration (Table 1). First, proteases were fractionated over a column of Sephadex G-25
to produce two peaks with 36.12 U and 1283.0 U. Of these, peak 2 (79.2% yield) showed greater proteolytic activity.
Next, peak 2 was additionally subjected to membrane ultrafiltration to produce fraction I with molecular mass
(MM) >50 kDa and fraction II with MM <50 kDa. Fraction II showed comparatively high proteolytic activity with 3.49 times
the purity and 36.5% yield.
The protein types and molecular-mass distributions of these SAP fractions were determined using gel electrophoresis
in polyacrylamide gel (PAAG, 15%) under dissociating conditions with sodium dodecyl sulfate (SDS, 0.02%). Figure 1
shows that the protease fractions contained many active proteins. SAPs after the two-step purification gave a significantly
stronger band with MM 14 kDa and much smaller amounts of other bands than commercial lamb abomasum active proteases
The MM of the peptides and the types of SAP were identified using LC-MS/MS. A comparison of the obtained
MS/MS spectra and observed peptides with spectra from the SWISS-PROT database showed that most peptides had stable
structures and were homologous to known active polypeptides (Table 2), including latent transforming growth factor, ribosomal
protein, albumin, etc. The results agreed with previously published data on the presence in sheep abomasum of active enzymes
such as growth factor and glycoprotein . SAPs also include previously unknown protein products requiring further studies
of their structure–function relationships.
1) Key Laboratory of Plants Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and
Chemistry, Chinese Academy of Sciences, Beijing South Road 40-1, Urumqi, 830011, Xinjiang, P. R. China; 2) State Key
Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, 830011, Urumqi, P. R. China, e-mail: firstname.lastname@example.org; 3) University of Chinese Academy of
Science, 100039, Beijing, P. R. China; 4) Educational and Experimental Center for High Technologies, Tashkent, 100174,
Uzbekistan, fax: (99871) 227 43 16. Translated from Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2018, pp. 443–445.
Original article submitted November 8, 2017.