Khan S M, Bolen W, Hargrave P A, Santoro M M, McDowell J H
Department of Medical Biochemistry, Southern Illinois University, Carbondale.
Eur J Biochem. 1991 Aug 15;200(1):53-9. doi: 10.1111/j.1432-1033.1991.tb21047.x.
Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.
已通过差示扫描量热法对来自牛的含视紫红质的视网膜杆状盘膜进行了检测。在67 mM磷酸盐pH 7.0的条件下，未漂白的杆状外段盘膜产生一个单一的主要吸热峰，变性温度（Tm）为71.9±0.4℃，热解链量热焓变（ΔHcal）为700±17 kJ/mol视紫红质。漂白后的杆状外段盘膜（在暴露于橙色光后在498 nm处失去吸光度的膜）产生一个单一的主要吸热峰，Tm为55.9±0.3℃，ΔHcal为520±17 kJ/mol视蛋白。无论是漂白的还是未漂白的杆状外段盘膜在热重扫时均未产生吸热峰。当在4-9的pH范围内检测热稳定性时，发现漂白的和未漂白的杆状外段盘膜的主要吸热峰在pH 6.1时显示出最大稳定性。观察到的漂白的和未漂白的杆状外段盘膜的ΔHcal值表现出膜浓度依赖性，在蛋白质浓度超过1.5 mg/mL时达到平稳。对于杆状外段盘膜的部分漂白样品，视蛋白的量热焓变似乎在某种程度上取决于漂白程度，表明膜内最近邻相互作用会影响视蛋白的解链。ΔHcal和Tm对于评估视紫红质从视蛋白再生的稳定性和完整性测试特别有用。其他因素，如样品制备和低浓度乙醇的存在，也会影响ΔHcal值，而Tm值保持相当恒定。这表明ΔHcal是监测视紫红质和视蛋白环境变化的敏感参数。
