Effect of protamine on the solubility and deamidation of human growth hormone

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• 作者：Elisabeth Ablinger^a ; ^b ; Stefan Wegscheider^b ; Walter Keller^c ; Ruth Prassl^d ; Andreas Zimmer^b ; ^{andreas.zimmer@uni-graz.at}
• 关键词：hGH ; human growth hormone ; pI ; isoelectric point ; NTU ; nephelometric turbidity unit ; DLS ; dynamic light scattering ; SAXS ; small-angle X-ray scattering ; ITC ; isothermal titration calorimetry ; CD ; circular dichroism ; RP-HPLC ; reversed-phase high-performance
• 刊名：International Journal of Pharmaceutics
• 出版年：2012
• 期刊代码：24_03785173
• 类别：pha
• 出版时间：10 May, 2012
• 卷：427
• 期：2
• 页码：209-216
• 文件大小：537 K

摘要

The effect of protamine on the solubility and deamidation of human growth hormone (hGH) was investigated. Protamine is an extremely basic peptide. It is isolated from sperm cells of salmon where it can build a complex with DNA due to electrostatic interactions. We hypothesize a similar electrostatic effect between negatively charged hGH and positively charged protamine residues. Arising cationic complexes are stabilized by electrostatic repulsion. This stabilizing complexation allows solubilization of hGH down to a pH of 5.4 (pI 5.3) at concentrations of 3.4 mg/ml. The minimal solubilizing molar ratio between hGH and protamine was found to be at least 1:23 (hGH:protamine) by turbidity and dynamic light scattering (DLS) measurements. Complexation was characterized by small-angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC). Electrostatic binding of protamine to hGH was also observed by a reversal in surface charge, as shown by zeta potential measurements. The presence of protamine did not alter the conformational structure of hGH which was determined by circular dichroism (CD) spectroscopy. A pH of 5.4 is known to slow deamidation of hGH and consequently retardation of hGH deamidation could be detected after 3 months storing by reversed-phase high-performance liquid chromatography (RP-HPLC).