光谱法研究细胞色素c氧化酶CuA结构域蛋白的稳定性

利用荧光光谱和圆二色谱研究了细胞色素c氧化酶CuA结构域蛋白的稳定性.结果表明,随着溶液pH值的升高,CuA结构域蛋白的稳定性降低.随着温度的升高,CuA结构域蛋白的二级结构遭到破坏.脱金属形式蛋白的热稳定性减小,表明双核金属铜中心的存在对CuA结构域蛋白的稳定性起重要作用.     

光谱法研究细胞色素C与proliNONOate之间的相互作用

细胞色素C(Cytochrome,Cyt c)作为细胞内线粒体的电子传递体,与NO之间的相互作用结果对于线粒体凋亡的检测有着重要的生物学意义。采用紫外-可见(UV-Vis)吸收光谱、电子顺磁技术(EPR)、时间过程光谱、圆二色(CD)光谱和同步荧光光谱等方法对处于不同价态的Cyt c与NO替代物,即proliNO/NO(proliNONOate)的相互作用过程,以及Cyt c在结合NO过程中蛋白空间结构的变化进行了详细研究。实验结果发现： 在模拟生理条件下,不同价态的Cyt c都能够直接与NO替代物(proliNO/NO)所产生的NO发生配位反应。推断出Cyt c与NO之间相互作用发生的可能机制： NO与Cyt c结合过程,是因为与Cyt c中Fe配位的轴向配体以及卟啉周围的取代基变化而发生结合的。具体反应过程为： 溶液中的NO诱导Cyt c中的Heme上Met80远离原来位置,Fe-S键断裂,进而空出的Fe与NO结合生成Fe-N键,从而生成新的Cyt c-NO配合物。研究表明： Cyt c-NO二元复合物不稳定,会发生光解离反应,通过线性拟合得出： 其解离过程属于准一级反应,解离速率为(0.071 1±0.039 6) s-1。同时,血红素Fe与NO间新键的形成,影响了色氨酸和酪氨酸微环境的变化;Cyt c二级结构受proliNO/NO浓度的影响,当proliNO/NO浓度低于8.6×10-4 mol·L-1时,Cyt c的α-螺旋特征峰强度变化很小,且位置不变;但proliNO/NO浓度高于8.6×10-4 mol·L-1时,即过量,Cyt c的二级结构变化很明显,说明过量的NO能导致Cyt c二级结构的破坏。NO与Cyt c配位反应机制的研究对于利用NO抑制线粒体内的氧气消耗,以及线粒体内NO的代谢具有重要的意义。     

Sensitive Spectrofluorimetric Determination of Cytochrome c with Spirocyclic Rhodamine B Hydrazide in Micellar Medium

A new spectrofluorimetric method for the determination of cytochrome c using spirocyclic rhodamine B hydrazide (RBH) as fluorogenic reagent in the presence of sodium dodecylbenzene sulfonate (SDBS) surfactant micelles was developed. The method was based on the reaction of cytochrome c with RBH, a colorless, nonfluorescent spirolactam of rhodamine B in SDBS micelles to give highly fluorescent rhodamine B and hence led to a large increase in fluorescence intensity. The dynamic range and detection limit for the determination of cytochrome c are 4.0–120 ng ml⁻¹ and 0.87 ng ml⁻¹ (3σ), respectively. The optimal conditions for the detection of cytochrome c were evaluated and the possible detection mechanism was also discussed.     

Interaction Between Cytochrome c and the Hapten 2,4-Dinitro-fluorobenzene by Electrospray Ionization Mass Spectrometry

采用电喷雾质谱、H/D交换和紫外光谱测定了半抗原2,4-二硝基氟苯和模型蛋白细胞色素c的相互作用. 电喷雾质谱结果表明,随着2,4-二硝基氟苯体积比百分含量的增加,细胞色素c的构象可以由天然的折叠构象转变为部分非折叠构象,最后转变为完全非折叠构象.细胞色素c在平衡态的H/D交换结合电喷雾质谱实验结果进一步确认了上述构象变化. 紫外光谱结果表明半抗原的加入对辅基血红素上的亚铁离子和细胞色素c上His18和Met80的强场配位作用没有明显的影响.     

Sonochemistry of cytochrome c. Evidence for superoxide formation by ultrasound in argon-saturated aqueous solution

Formation of superoxide anion radicals (O₂^·−) induced by 50 kHz ultrasound in argon (Ar)-saturated aqueous solution was studied. Although EPR-spin trapping study with 5,5-dimethyl-1-pyrroline N-oxide (DMPO;100 mM) revealed the formation of DMPO-adducts of hydroxyl radicals (^·OH) and hydroperoxy radicals (HO₂^·; acid form of O2^·−) in O₂-saturated solution after sonication, no evidence of HO₂^· was found in Ar-saturated solution. When ferricytochrome c (cytochrome c) in Ar-saturated aqueous solution was sonicated, the reduced form of cytochrome c was observed and 80% of its formation was inhibited by the addition of superoxide dismutase (SOD). Sodium formate enhanced the production of the reduced form of cytochrome c. The % inhibition by SOD for the reduction increased in the order of Xe > Ar > He in accord with the higher temperatures of the cavitation bubbles. These results indicate that the O₂^·− is formed by directly by the sonolysis of water in the absence of O₂ when the temperature of cavitation bubble collapse is sufficiently high.     

Aging Thyroarytenoid and Limb Skeletal Muscle: Lessons in Contrast

Voice production is vital throughout life because it allows for the communication of basic needs as well as the pursuit and enjoyment of social encounters. Unfortunately, for many older individuals the ability to produce voice is altered. Structural and functional declines in the neuromuscular system occur with aging and likely contribute to the modification of voice. One specific target of the aging process is the thyroarytenoid (TA) muscle, the primary muscle of voice production. The objectives of this overview article are to (1) share current findings related to the aging of limb skeletal muscle, (2) identify age-related morphological and physiological features of TA muscle, (3) compare and contrast age-related changes in TA with those in limb skeletal muscle, and (4) describe therapies for reversing sarcopenia in limb muscle and consider the applicability of these therapies for addressing vocal fold atrophy and age-related voice changes. The article shares current knowledge from the basic sciences related to skeletal muscle aging and compares/contrasts typical muscle aging to TA aging. Current evidence suggests that (1) the TA muscle undergoes notable remodeling with age, (2) aging of the TA is multifactorial, resulting from a myriad of neurologic, metabolic, and hormonal changes, many of which are distinct from the age-related processes of typical limb skeletal muscle, (3) investigation of the aging of the TA and its role in the aging of voice is in its infancy, and (4) potential behavioral and nonbehavioral therapies for reversing aging of the TA must be further examined.     

pH-Induced Changes in the Secondary Structure of Cytochrome c: an Infrared Spectroscopic Study

The infrared spectra of 3mM solutions of horse heart cytochrome c were recorded as a function of pD(corresponding to acid high spin form, acidic low spin form, native form, lysine form, the so-termed “strained lysine form” as well as the so-termed “A state” of cytochrome c). An analysis of the pH-induced changes in the secondary structure was performed based on changes in the conformation-sensitive amide I bands of this protein. In lysine and strained lysine forms, the contents of random structure increase at the expense of α-helix. In acidic low. spin form (pH4–2.5), cytochrome c was almost unfolded. The proportion of α-helix had a substantial decrease while the contents of both random structure and 3₁₀-helices got a rise. In acidic high spin form (when pH is below 2.5), a further decrease of pD gave no rise to a continued unfolding in cytochrome c but made it refold to the “A state” with properties similar to those of a molten globular state. Adding enough KC1 to the cytochrome c solution(pD2.2 with 0.5MKC1 and pD3.0 with 1.5 MKCl) in which cytochrome c was nearly fully unfolded also led to the formation of A state of cytochrome c. The compositions of all types of secondary structures in A state were evaluated. The behavior of 3₁₀-helices in acidic high/low spin form indicated they might be intermediates between α-helices and random structure in acidic solution as proposed by Miick et al.     

基于磁共振的胞浆中无标记酵母细胞色素c构象变化追踪

细胞色素c(cytochrome c,cyt c)是一个重要的多功能蛋白.在线粒体中,它作为载体传递电子.而在细胞质中,它可能会作为凋亡起始因子启动细胞凋亡程序.复杂的细胞质环境是否会对其构象产生影响,以及产生怎样的影响,目前仍然没有得到确证.本研究通过无标记的基于甲基的核磁共振（nuclear magnetic resonance,NMR）技术追踪了野生型酿酒酵母iso-1 cyt c在酵母细胞匀浆液中的构象变化.发现cyt c在细胞匀浆中至少存在4种不同的氧化态构象和1种还原态构象.而且随着时间的推进,不同构象之间发生转换.结果表明cyt c的构象会随环境改变,这可能与抵抗氧化应激相关.     

A switch in the electron transfer from heme a to binuclear centre of cytochrome c oxidase

New experimental evidence that a switch controls the reduction of the heme a₃－Cu_B binuclear centre has been observed in the N₂-dried thin film of purified cytochrome oxidase. When immersing the enzyme film into the acid phosphate buffer with extremely low concentration of dithionite, a spectrum was given to show a reduction of heme a with no electrons resting on Cu_A. By increasing dithionite, electrons could be accumulated gradually on Cu_A, but the binuclear centre still remains in the oxidized state. When the accumulation of electrons on Cu_A and/or heme a exceeded a threshold, a turnover of reduction of the binuclear centre and oxidation of heme a occurred abruptly. This switch-like action is pH-dependent.     

O2 binding to cytochrome c oxidase-inspired nanomaterials

We investigate O₂ binding to cytochrome c oxidase (CcO)-inspired nanomaterials using ab initio density functional calculations. We consider iron-porphyrin (FeP) and copper-(imidazole)₃ [Cu(Im)₃] as a representative of the active binuclear center, and explore the effect of the Cu(Im)₃ on the FeP-O₂ adduct in relation to the geometric, vibrational, electronic and energetic properties. We find that the Cu(Im)₃ induces the weak O-O and Fe-O bonds mainly because of the electron transfer to the O₂ and the spin polarization of the Fe and O₂ by bridging the O₂ between the Fe and Cu, possibly resulting in the facile O₂ dissociation.     

Quantitative Evaluation of Oxygenation and Metabolism in the Human Skeletal Muscle

The forearm muscles of five healthy males were monitored for changes in microvessel hemoglobin saturation (SO_2-TRS) by near infrared time-resolved spectroscopy (NIR_TRS) and changes in phosphorus metabolites by magnetic resonance spectroscopy (³¹P-MRS) during 12 min of resting arterial occlusion. Muscle oxygenation and phosphorus metabolites were also measured during grip exercises at varying intensities. Upon the initiation of occlusion, SO_2-TRS fell progressively until it reached a plateau in the latter half of the occlusion. Phosphocreatine (PCr) began to decrease around 6 min after the initiation of arterial occlusion. The resting O₂ store and O₂ consumption were 295 μM and 0.95 μM/sec, respectively-values which reasonably agree with the reported results. A significant correlation was observed between the changes in SO_2-TRS and PCr during exercise (r² = 0.80, p < 0.001). These results indicate that NIR_TRS is able to provide reliable information about resting metabolism and oxidative rate during exercise. NIR_TRS and MRS are useful to monitor oxygenation and energetics noninvasively in the human muscle.