Separovic F, Lam Y H, Ke X, Chan H K
School of Chemistry, University of Melbourne, Parkville, Vic, Australia.
Pharm Res. 1998 Dec;15(12):1816-21. doi: 10.1023/a:1011993620177.
The mobility of protein in powders at different hydration levels was studied in relation to aggregation and activity.
Magic angle spinning 13C, 15N, 1H, 2H, and 17O NMR techniques were used to determine changes in the mobility of surface residues in proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (omega0, T1) and low frequency (omega1,T1p) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays.
There was little change in T1p observed with increasing humidity. The results show, however, that there is a decrease in T1 for DNase, insulin and lysozyme at relative humidities ranging from 0-98%, and we propose that the reduction in T1 is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined directly using 17O NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data.
Mobility of protein molecules was determined by solid-state NMR over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of proteins were strongly correlated to change in molecular mobility.
研究不同水合水平下蛋白质在粉末中的流动性与聚集及活性的关系。
使用魔角旋转13C、15N、1H、2H和17O NMR技术来确定蛋白质表面残基流动性随水合作用的变化，并与活性变化相关联。对储存在不同相对湿度下的冻干脱氧核糖核酸酶、胰岛素和溶菌酶进行了高频（ω0，T1）和低频（ω1，T1p）运动的NMR弛豫测量。通过高效尺寸排阻色谱法和生物测定法测定冻干蛋白质的水分诱导聚集和酶活性。
随着湿度增加，T1p几乎没有变化。然而，结果表明，在相对湿度为0 - 98%的范围内，脱氧核糖核酸酶、胰岛素和溶菌酶的T1降低，我们认为T1的降低与不同湿度下储存期间蛋白质的聚集敏感性有关。使用17O NMR实验直接测定了水的流动性。我们发现，随着弱结合水含量的增加，蛋白质表面流动性降低，并伴随着聚集增加。不同湿度下的聚集测量结果与溶菌酶的生物测定结果相关，并且与水合数据一致。
通过固态NMR在广泛的相对湿度范围内测定了蛋白质分子的流动性，发现含水量导致蛋白质分子流动性发生变化。蛋白质的聚集和活性与分子流动性的变化密切相关。
