title: Preparation, structural analysis and bioactivity of ribonuclease A-albumin conjugate: tetra-conjugation or PEG as the linker.
abstract: Ribonuclease A (RNase A) is a therapeutic enzyme with cytotoxic action against tumor cells. Its clinical application is limited by the short half-life and insufficient stability. Conjugation of albumin can overcome the limitation, whereas dramatically decrease the enzymatic activity of RNase A. Here, three strategies were proposed to prepare the RNase A-bovine serum albumin (BSA) conjugates. R-SMCC-B (a conjugate of four RNase A attached with one BSA) and R-PEG-B (a mono-conjugate) were prepared using Sulfo-SMCC (a short bifunctional linker) and mal-PEG-NHS (a bifunctional PEG), respectively. Mal-PEG-NHS and hexadecylamine (HDA) were used to prepare the mono-conjugate, R-HDA-B, where HDA was adopted to bind BSA. The PEG linker can elongate the proximity between RNase A and BSA. In contrast, four RNase A were closely located on BSA in R-SMCC-B. R-SMCC-B showed the lowest K(m) and the highest relative enzymatic activity and k(cat)/K(m) in the three conjugates. Presumably, the tetravalent interaction of RNase A in R-SMCC-B can increase the binding affinity to its substrate. In addition, the slow release of BSA from R-HDA-B may increase the enzymatic activity of R-HDA-B. Our study is expected to provide strategies to develop protein-albumin conjugate with high therapeutic potential.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Chunju
authors.initials: C
authors.lastname: Li
authors.forename: Qixun
authors.initials: Q
authors.lastname: Lin
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Lijuan
authors.initials: L
authors.lastname: Shen
authors.forename: Guanghui
authors.initials: G
authors.lastname: Ma
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
mesh_terms: D000818:Animals; D002417:Cattle; D016503:Drug Delivery Systems / Q000379:methods*; D008027:Light; D008958:Models, Molecular; D011092:Polyethylene Glycols / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D012542:Scattering, Radiation; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
chemical_list: D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D012259:Ribonuclease, Pancreatic