PubMed · 2314269
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M P Deutscher. 1990. Maintaining protein stability.. https://doi.org/10.1016/0076-6879(90)82010-y
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The history of the regulatory control of pharmaceutical ingredients is briefly reviewed. The impact of legislation, its interpretation through European and international guidelines and resulting licensing policies are discussed. The important role played by excipients in modern formulations is recognised and some examples are given of the unexpected consequences of inappropriate choices. Some data are provided on the extent to which excipients and active ingredients appear in UK-licensed medicines and on the reference standards (pharmacopoeial and non-official) used to control the excipients.
Using site-directed mutagenesis, His(143) on the alpha-helix F of PAI-1 was substituted by Lys, Asp, Phe and Thr, respectively. The generated single-site changed plasminogen activator inhibitor-1 (PAI-1) mutants were expressed in Escherichia coli and purified by heparin-Sepharose and anhydrotrypsin agarose chromatographies. When compared with wild-type (wtPAI-1), the PAI-1 mutants His143Asp and His143Phe had shorter half-lives at pH 7.5 (1.1 and 1.4 h, respectively, vs. 2 h for wtPAI-1). They also exhibited less pH dependency of their stability, with half-lives at pH 5.5 of 2.5 and 2.2 h, respectively, vs. 18 h for wtPAI-1. However, the PAI-1 mutants His143Lys and His143Thr had similar properties as wtPAI-1 in this respect. In conclusion, our results suggest that His(143) in one way or another might be involved in the pH-dependent stability of PAI-1. However, it seems that the protonation of this particular residue is of less importance. The PAI-1 mutants His143Asp and His143Phe only displayed about 20% of the specific activity obtained for wtPAI-1, because they, to a large extent, act as substrates for tissue-type plasminogen activator.