Kinetics and thermodynamics of oxygen and carbon monoxide binding to the T-state hemoglobin of Urechis caupo.
Kinetics and thermodynamics of oxygen and carbon monoxide binding to the T-state hemoglobin of Urechis caupo.
复制标题
氧和一氧化碳与 Urechis caupo T 态血红蛋白结合的动力学和热力学。
DOI：
10.1021/bi00476a012
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Parkhurst,LJ
中科院分区：
文献类型：
--
作者：
Martin,KD;Parkhurst,LJ
Please try later.
Materials and MethodsPreparation of Hb. Specimens of Urechis caupo were ob-tained from Sea Life Supply, Sand City, CA. Animals were bled by dorsal incision, the coelomic fluid (50 mL) was cen-trifuged at 4000g at 4 C for 10 min, and the packed cells were washed two times with 1% NaCl with centrifugation after each washing. Lysing was achieved by subjecting the cells to three freeze-thaw cycles in 0.10 Mkp¡(20-mL total volume) at pH 6.0. Cell debris was removed by centrifugation at 27000g for 30 min. The solution was diluted to 40 mL with 0.10 M KPj, 0.10 g of celite was added with stirring, and the suspension was then centrifuged at 27000g for 30 min. The Hb was stored frozen, as droplets, in liquid nitrogen. Kinetic studies run on freshly prepared Hb showed functionality identical with that of the liquid nitrogen stored protein. In one study, the hem-oglobin was fractionated on a 2.2 cm X 12 cm. column ofCMC-50 Sephadex (Pharmacia) equilibrated with 0.02 M potassium phosphate, pH 6. The minor band eluted under these conditions; then, the major component was isolated by elution with 0.05 M Tris-HCl, pH 8.5. Instrumentation and Data Analysis. The stopped-flow apparatus has been described elsewhere (Parkhurst et al., 1980). Data were acquired from both rapid flow and laser photolysis initiated reactions by a Computerscope Model ISC-16 (RC Electronics, Santa Barbara, CA) in an IBM PC-XT microcomputer. The data (typically 1000 points per run) were then digitally filtered with third-order polynomial smoothing (Savitzky & Golay, 1964) and least-squares fit to both one-and two-exponential models. For none of the re-actions were the biexponential fitssignificantly better than for a single-exponential fit. For CO dissociation, a small stopped-flow apparatus was built into the Cary 210 spectro-photometer, with the cuvet situated in a thermostated brass block having three optical ports. The cuvet assembly was modified to include flow solution reservoirs, which were thermostated in the block along with the reaction chamber. A thermistor was embedded immediately adjacent to the reservoirs to monitor temperature. A high-energy photographic flash tube (Wabash Electroflash, type Rl 140, Wa-bash Corp., Brooklyn, NY) was situated in the Cary at the third opticalport, perpendicular to the monitoring beam, and was used for photolysisto drive the CO dissociation reactions to completion after approximately three half-times. The Cary was interfaced to an Apple II+ microcomputer for storage of absorbance vs time data. For laser photolysis, a Phase-R dye laser (New Durham, NH), tube ModelDL-18Y, was used with Coumarin 540A dye (Exciton Chemical Co., Inc., Day-ton, OH). The cuvet was situated in a thermostated brass block with four optical ports and had a thermistor placed just above the path of the monitoring beam. It was convenient to use the regular stopped-flow instrument for the quantum yield studies, since the HbCO and MbCO1 solutions could be al-ternately placed in the cell, and the closed system ensured that no gas exchangeoccurred during the course of the experiment. An argon ion laser (Model 75, Lexel, Palo Alto, CA) was used in the quantum yield studies. The beam was dispersed by a lens onto one end of an optical fiber bundle, which terminated in a rectangular array directly over the stopped-flow cuvet and