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. 2015 Mar 4;15:60. doi: 10.1186/s12866-015-0394-8

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© Kozik et al.; licensee BioMed Central. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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HPLC/MS characteristics of the Sap-catalyzed cleavage of the HK-D4 peptide. Ten μM of the synthetic peptide HK-D4 (which has the ISLMKRPPGFSPFRSSRIGEIKEET amino acid sequence) were treated with recombinant Sap1–10 in citrate (50 mM) or phosphate buffers (25 mM) at the optimal pH for the general proteolytic activity [26] of each individual Sap (specified in the figure) at an enzyme:substrate molar ratio of 1:50 for 24 hours at 37°C. The reaction was stopped using HCl, and the samples were analyzed using reversed-phase HPLC on an Eurosil Bioselect 300–5 C-18 column (Knauer) in a TFA-water-ACN binary gradient system. The fractions, which were collected at the major absorbance peaks (215 nm), were evaporated and analyzed using ESI-MS/MS in order to determine their amino acid sequence.