Chemical modification of recombinant hirudin with palmitic acid in mixed aqueous-organic solutions. (February 2019)

• Record Type: Journal Article
• Authors: Wang, Shu-Chang; Wang, Xu-Dong; Teng, Xin-Nan; Xiu, Zhi-Long
• Is Part Of: Process biochemistry. Volume 77(2019)
• Page Start: 85
• Page End: 92
• Publication Date: 2019-02
• Subjects: Recombinant hirudin -- Chemical modification -- Fatty acid-conjugation -- Long-chain fatty acids -- Palmitic acid -- Mixed aqueous-organic solutions; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2018.11.022
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: Recombinant hirudin variant-2 (HV2) was conjugated with palmitic acid (PAL). The selectivity of this reaction was improved in mixed aqueous-DMSO solution. Three mono-PAL-HV2 isomers was efficiently separated by prep-HPLC. Mono-PAL-HV2-3 showed the highest selectivity and the highest in vitro bioactivity. The modification site of mono-PAL-HV2-3 was identified as Lys 27 of HV2. Abstract: In this study, chemical modification of recombinant hirudin variant-2 (HV2) with palmitic acid (PAL) was proposed as an alternative approach to circumvent the limitations of PEGylation. To facilitate a sufficient contact of the hydrophilic HV2 to the hydrophobic PAL, thereby improving the reaction specificity to achieve the desired mono-PAL-HV2 with high retained bioactivity, the reaction was developed in mixed aqueous-organic solutions. Compared with HV2 conjugation with PAL-benzotriazole (PAL-BTA) in mixed aqueous-NMP solution, the conjugation of HV2 with PAL- N -hydroxysuccinimide (PAL-NHS) in mixed aqueous-DMSO solution could improve the site-specific conjugation of one PAL molecule to a particular lysine residue. Furthermore, the reaction mixture of the latter was further purified by preparative liquid chromatography. Three mono-PAL-HV2 isomers were obtained and retained 36%, 4% and 89% of the in vitro anticoagulant activity of unmodified HV2, respectively. One of the mono-PAL-HV2 isomers, namely, mono-PAL-HV2-3, was isolated with the highest selectivity and exhibited the highest in vitro anticoagulant bioactivity. Modification site analysis of mono-PAL-HV2-3 revealed that a single PAL molecule was conjugated at Lys27 of HV2. This study presented a successful PAL modification in which site-specific reaction was improved to achieve the desired mono-PAL-HV2 with highly retained bioactivity in mixed aqueous-organic solutions.