Protein photolysis kinetics

The kinetics of reactions of NO with ferri- and ferro-heme proteins and models under ambient conditions have been studied by time-resolved spectroscopic techniques. Representative results are summarized in Table I (22-28). Equilibrium constants determined for the formation of nitrosyl complexes of met-myoglobin (metMb), ferri-cytochrome-c (Cyt111) and catalase (Cat) are in reasonable agreement when measured both by flash photolysis techniques (K= konlkQff) and by spectroscopic titration in aqueous media (22). Table I summarizes the several orders of magnitude range of kon and kQs values obtained for ferri- and ferro-heme proteins. Many k0f[ values were too small to determine by flash photolysis methods and were determined by other means. The small values of kQ result in very large equilibrium constants K for the... 

Recent advances in measuring the kinetics of the various electron-transfer steps in this system have been achieved by use of flash photolysis of ruthenated derivatives of cytochrome c (Ru-Cc) (17-19). In these studies [Ru(bpy)3]2+ is covalently bound to a surface residue at a site that does not interfere with the docking of cytochrome c to cytochrome c oxidase. Solutions are then prepared containing both Ru-Cc and cytochrome c oxidase, and the two proteins associate to form a 1 1 complex. Flash photolysis of the solution leads directly to the excitation of the RuII(bpy)3 site, which then reduces heme c very rapidly. This method thus provides a convenient means to observe the subsequent intracomplex electron transfer from heme c to cytochrome c oxidase and further stages in the process. 

The kinetics of myoglobin oxidation and reduction have been studied by a variety of experimental techniques that include stopped-flow kinetics, pulse radiolysis, and flash photolysis. In considering this work, attention is directed first at studies of the wild-type protein and then at experiments involving variants of Mb. 

Smface modification with ruthenium complexes has proven valuable in studies of both interprotein and intraprotein electron transfer in systems that are difflcult to stndy by traditional kinetic tools. The choice of ruthenium complexes in these investigations stems from an extensive photochemistry as well as exceptional thermal stability. The photochemistry provides a means of examining reactions over a time range of nanoseconds to seconds by laser-flash photolysis and the thermal stability allows researchers to covalently bind a wide variety of complexes to proteins with... 

UV photolysis of CpMn(CO)3 in toluene leads to loss of CO and formation of CpMn(CO)2( ] -toluene). Kinetic studies suggest that the binding energy of the toluene is ca. 60kJmor. The binding of H2 to CpMn(CO)2 has been studied in supercritical CO2 solvent. It has been proposed that pyrylium and pyridinium salts such as (35) can be used to label proteins and thereby aid in the detection and characterization of receptor sites. Cymantrene bound to lysine residues of bovine serum albumin (BSA) has been used as a redox label. Electrochemical reduction of the label established an impressive BSA detection limit of 2 x 10 M. 

This paper will concentrate on three areas of photolysis where important questions concerning biological systems are addressed. The first will be the well-established but still vital area of the heme proteins that have photolabile ligands. The second area will be with the use of triplet probes on proteins and DNA. The third area covered will be those special proteins that are photochemically active. To avoid making technology take precedence over science, many experimental details will be discussed in the process of our attempt to provide a unifying thread to recent trends in biochemical photolysis. The authors will not attempt to discuss the various aspects of bimolecular kinetics. We will instead stress the aspects of photolysis where the protein is believed to play an important role in the overall kinetic picture. 

Kerrick WGL, Hoar PE (1981) Inhibition of smooth muscle tension by cyclic AMP-de-pendent protein kinase. Nature 292 253-255 Khromov A, Somlyo AV, Trentham DR, Zimmermann B, Somlyo AP (1995) The role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle a flash photolysis study. Biophys J 69 2611-2622 Khromov AS, Somlyo AV, Somlyo AP (1996) Nucleotide binding by actomyosin as a determinant of relaxation kinetics of rabbit phasic and tonic smooth muscle. J Physiol (Lond) 492 669-673... 

---