Vapor pressure osmometry studies of osmolyte-protein interactions: Implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro
Vapor pressure osmometry studies of osmolyte-protein interactions: Implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro
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DOI：
10.1021/bi992887l
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发表时间：
2000-04-18
期刊：
影响因子：
2.9
通讯作者：
Record, MT
中科院分区：
文献类型：
--
作者：
Courtenay, ES;Capp, MW;Record, MT
为了解释或预测生物聚合物过程在体内和体外对溶质浓度变化和水活度（渗透压）耦合变化的响应，需要定量了解溶质和水与生物聚合物表面相互作用的热力学后果。为此，我们报告等渗优先相互作用系数（伽马（mu 1））确定的蒸汽压渗透压法（VPO）在很宽的浓度范围内的天然牛血清白蛋白（BSA）和六个小溶质之间的相互作用。这些包括大肠杆菌胞质渗透物[谷氨酸钾（K（+）Glu（-）），海藻糖]，大肠杆菌。大肠杆菌芽孢杆菌保护剂（脯氨酸、甘氨酸甜菜碱），以及甘油和N-氧化三甲胺（TMAO）。对于所有六种溶质，γ（mu 1）和相应的透析优先相互作用系数γ（mu 1，mu 3）（均根据VPO数据计算）为负值; γ（mu 1，mu 3）与至少高达1 oz（摩尔）的本体溶质质量摩尔浓度（m（3）（本体））成比例。γ（mu 1，mu 3）的负值表示在透析平衡时优先将这些溶质从BSA溶液中排除，并对应于这些溶质在BSA附近的局部浓度，其低于其本体浓度。在所研究的溶质中，甜菜碱是最不被排除的（γ（mu 1，mu 3）/m（3）（本体）= -49 +/- 1 m（-1））;甘油是最不被排除的（γ（mu 1，mu 3）/m（3）（本体）= -10 +/- 1 m（-1））。在这些极端值之间，γ（mu 1，mu 3）/m（3）（本体）3 3的大小以甘氨酸甜菜碱>>脯氨酸>TMAO >海藻糖近似于K+Glu- >甘油的顺序减小。大肠杆菌渗透调节物质从BSA表面被排除的顺序与它们作为渗透调节剂的有效性相关，渗透调节剂可增加大肠杆菌的生长速率。大肠杆菌在高外部渗透压。对于最排除的溶质（甜菜碱），Gamma（mu 1，mu 3）提供了天然BSA水合作用的最小估计值，约为2.8 x 10（3）H2O/ BSA，相当于略低于单层（估计与3.2 x 10（3）H2O相似）。因此，在这里研究的溶质，只有甜菜碱可能是适合用于在体外渗透应力实验作为一个直接的探针，以量化在生物聚合物过程中的蛋白质表面的水合作用的变化。然而，更一般地说，我们的结果和分析导致的建议，这些溶质中的任何一种都可以用来量化的水可及表面积（阿萨）在生物聚合物过程中的变化，一旦优先的相互作用的溶质与生物聚合物表面被适当地考虑。
TO interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu 1)) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu 1) and the corresponding dialysis preferential interaction coefficient Gamma(mu 1,mu 3) (both calculated from the VPO data) are negative; Gamma(mu 1,mu 3) is proportional to bulk solute molality (m(3)(bulk)) at least up to 1 oz (molal). Negative values of Gamma(mu 1,mu 3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu 1,mu 3)/m(3)(bulk) = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu 1,mu 3)/m(3)(bulk) = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu 1,mu 3)/m(3)(bulk) 3 3 decreases in the order glycine betaine >> proline >TMAO > trehalose approximate to K+Glu- > glycerol. The order of exclusion of E, coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu 1,mu 3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H2O/ BSA, which corresponds to slightly less than a monolayer (estimated to be similar to 3.2 x 10(3) H2O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.