Chan H K, Ongpipattanakul B, Au-Yeung J
Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080, USA.
Pharm Res. 1996 Feb;13(2):238-42. doi: 10.1023/a:1016091030928.
To determine if a protein changes when it is compressed into a KBr pellet for FTIR spectroscopy measurement in the solid state, using recombinant human deoxyribonuclease I (rhDNase) as an example.
Lyophilized rhDNase with KBr compressed at different pressures were analyzed by FTIR spectroscopy, size exclusion HPLC and enzymatic activity assay. Different protein/KBr weight ratios and residual water contents were studied for their possible effects on aggregation.
Depending on the pressure, a loss of enzymatic activity accompanied by an increase in soluble high molecular weight aggregates of the protein (up to approximately 15%) was demonstrated. Aggregation was reduced to less than 5% by a suitable dilution of the protein in KBr (1 in 1000). In contrast, water content variability (1-11 wt. %) did not affect aggregation.
The findings emphasize the importance to examine for protein integrity when using the KBr method for FTIR sample preparation. Protein aggregation may be minimized by optimizing the sample preparation condition such as changing the protein/KBr weight ratio.
以重组人脱氧核糖核酸酶I（rhDNase）为例，确定蛋白质在被压制成用于固态傅里叶变换红外光谱（FTIR）测量的溴化钾（KBr）压片时是否发生变化。
采用FTIR光谱、尺寸排阻高效液相色谱法和酶活性测定法，对在不同压力下与KBr一起压缩的冻干rhDNase进行分析。研究了不同的蛋白质/KBr重量比和残余含水量对聚集的可能影响。
根据压力不同，蛋白质的酶活性丧失，同时可溶性高分子量聚集体增加（高达约15%）。通过在KBr中适当稀释蛋白质（1:1000），聚集率降低至5%以下。相比之下，含水量变化（1 - 11重量%）不影响聚集。
研究结果强调了在使用KBr法进行FTIR样品制备时检查蛋白质完整性的重要性。通过优化样品制备条件，如改变蛋白质/KBr重量比，可使蛋白质聚集最小化。
