共查询到20条相似文献,搜索用时 31 毫秒
1.
Andrea Mahn Alejandro Reyes Mauricio Zamorano Wildo Cifuentes Maritza Ismail 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(15-16):1038-1044
The proteomic analysis of plasma is extremely complex due to the presence of few highly abundant proteins. These proteins have to be depleted in order to detect low abundance proteins, which are likely to be of biomedical interest. In this work it was investigated the applicability of hydrophobic interaction chromatography (HIC) as a plasma fractionation method prior to two-dimensional gel electrophoresis (2DGE). The average hydrophobicity of the 56 main plasma proteins was calculated. Plasma proteins were classified as low, medium and highly hydrophobic through a cluster analysis. The highly abundant proteins showed a medium hydrophobicity, and therefore a HIC step was designed to deplete them from plasma. HIC performance was assessed by 2DGE, and it was compared to that obtained by a commercial immuno-affinity (IA) column for albumin depletion. Both methods showed similar reproducibility. HIC allowed partially depleting α-1-antitrypsin and albumin, and permitted to detect twice the number of spots than IA. Since albumin depletion by HIC was incomplete, it should be further optimized for its use as a complementary or alternative method to IA. 相似文献
2.
A method of affinity chromatography based on the trapping of actin filaments within agarose gel beads is described. This method can be used for the purification of myosin and its active proteolytic subfragments, as well as for studies on the interaction between actin and these proteins. Actin columns stabilized by phalloidin bind myosin, heavy meromyosin (HMM), and heavy meromyosin subfragment 1 (HMM-S1) specifically and reversibly. The effect of pyrophosphate and KCl on the dissociation of actomyosin, acto-HMM, or acto-HMM-S1 complex is reported. We also describe the single-step purification of myosin from a crude rabbit psoas muscle extract. 相似文献
3.
Functional association of essential splicing factor(s) with PRP19 in a protein complex. 总被引:11,自引:1,他引:10
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W Y Tarn C H Hsu K T Huang H R Chen H Y Kao K R Lee S C Cheng 《The EMBO journal》1994,13(10):2421-2431
We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs. It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly. We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing. Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affinity chromatography. The isolated complex is functional for complementation for the heat-inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19. At least three of these can interact directly with the PRP19 protein. We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins. 相似文献
4.
For many protein multimers, association and dissociation reactions fail to reach the same end point; there is hysteresis preventing one and/or the other reaction from equilibrating. We have studied in vitro assembly of dimeric hepatitis B virus (HBV) capsid protein and dissociation of the resulting T = 4 icosahedral capsids. Empty HBV capsids composed of 120 capsid protein dimers were more resistant to dissociation by dilution or denaturants than anticipated from assembly experiments. Using intrinsic fluorescence, circular dichroism, and size exclusion chromatography, we showed that denaturants dissociate the HBV capsids without unfolding the capsid protein; unfolding of dimer only occurred at higher denaturant concentrations. The apparent energy of interaction between dimers measured in dissociation experiments was much stronger than when measured in assembly studies. Unlike assembly, capsid dissociation did not have the concentration dependence expected for a 120-subunit complex; consequently the apparent association energy systematically varied with reactant concentration. These data are evidence of hysteresis for HBV capsid dissociation. Simulations of capsid assembly and dissociation reactions recapitulate and provide an explanation for the observed behavior; these results are also applicable to oligomeric and multidomain proteins. In our calculations, we find that dissociation is impeded by temporally elevated concentrations of intermediates; this has the paradoxical effect of favoring re-assembly of those intermediates despite the global trend toward dissociation. Hysteresis masks all but the most dramatic decreases in contact energy. In contrast, assembly reactions rapidly approach equilibrium. These results provide the first rigorous explanation of how virus capsids can remain intact under extreme conditions but are still capable of "breathing." A biological implication of enhanced stability is that a triggering event may be required to initiate virus uncoating. 相似文献
5.
Organisms have harnessed the unique chemistry of copper for a variety of purposes. However, that same chemistry makes this essential metal toxic at elevated concentrations. Metallothioneins (MTs), a family of small metal-binding proteins, are thought to play a crucial role in the regulation of this reactive ion. Here we report that copper-metallothioneins from the American lobster, Homarus americanus, interact with the tripeptide glutathione (gamma-Glu-Cys-Gly). Glutathione in the cytosolic fraction prepared from the digestive gland of the American lobster coelutes with copper-metallothionein during size-exclusion chromatography. The latter protein can be separated into three isoforms by anion-exchange chromatography. All three isoforms belong to the class I MTs. CuMT-I and -II are very similar, whereas CuMT-III is distinct from isoforms I and II. The interaction between glutathione and MT isoforms was examined by ultrafiltration experiments and size-exclusion HPLC. CuMT-III forms a stable 1:1 complex with glutathione, with a dissociation constant of 1 microM. CuMT-I/II makes a transient complex with glutathione, which releases copper as a copper-glutathione complex. This complex can function as the source of Cu(I) in the restoration of the oxygen-binding capacity of copper-free hemocyanin. These studies suggest that metallothionein and glutathione are intricately linked in the biochemistry of copper regulation. 相似文献
6.
Gruen M Becker C Beste A Reinstein J Scheidig AJ Goody RS 《Protein science : a publication of the Protein Society》1999,8(11):2524-2528
The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2'-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2'-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2'-halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2'-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras(Y32W) (fluorescent mutant) and RabS. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of approximately 100 (p21ras) or approximately 10(5) (Rab5), and the resulting equilibrium dissociation constants are in the nm or microM range, respectively. The data demonstrate that 2'halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 10(5)-fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the microM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex. 相似文献
7.
Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications. 相似文献
8.
Two electrophoretically distinguishable species of the 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis are detectable by standard polyacrylamide gel electrophoresis in the absence of urea, detergents, or any other protein-denaturing reagents. The slower species (type IA) can be converted into the faster species (type IB) by treatment with ATP, and the fast form converts into the slow form when aged at 4 degrees. The enzyme undergoes these conversions both when it is free in solution and when it is membrane bound. The ATP analog adenylyl imidodiphosphate (AMP-PNP) gives the conversion without being hydrolyzed and without causing any apparent change in the mass of the protein, which suggests that the conversion may be a ligand-induced conformational change. Types IA and IB can convert into three other electrophoretically distinguishable species (types IIA, IIB, and III) if the purification procedure involves chromatography on a DEAE-Sephadex column equilibrated in phosphate buffer. These conversions can be prevented if the column is eluted in morpholinoethanesulfonic acid (Mes) buffer and KCl. Type IIA is convertible into type IIB by ATP treatment. Types IA and IB will also convert into types IIA and IIB and finally into type III when aged for extended periods of time at 4 degrees, without a detectable change in mass. Coupling factor activity is lost when type I enzyme converts into type II enzyme, as is the ability of the enzyme to bind to the membrane. However, ATPase activity does not change significantly. The mitochondrial 13S coupling factor shows up to three electrophoretically distinguishable species. The use of phosphate buffer during DEAE-Sephadex chromatography gives conversion of slower species into faster species. ATP treatment does not give interconversions, and aging at 4 degrees gives only a slow dissociation of the enzyme into subunits. The chloroplast 13S coupling factor also shows up to three electrophoretic species. Incubation with ATP does not give interconversions, but a temperature-dependent conversion of the major species into a faster species occurs upon aging. The subunit composition of the three 13S enzymes is very similar by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the major difference being in the number of classes of small polypeptides. 相似文献
9.
A method of fractionation of hnRNP constituents adaptable to large-scale preparation is presented. It is based on differential resistance to salt dissociation of the two classes of units of hnRNP, the 30--50S monoparticles and the heterogeneous complexes. The monoparticle proteins were released from hnRNP by 0.4 M NaCl. They were separated from the salt-resistant RNP corresponding to the heterogeneous complexes in three steps: chromatography on DEAE-cellulose, high-speed centrifugation, and Bio-Gel chromatography. The latter chromatography permitted a first fractionation of monoparticle proteins according to molecular weight. Such fractions may serve for purification of individual proteins of molecular weight below 80 000. After the two first steps, two fractions of salt-resistant RNP were obtained. In addition to heterogeneous RNA up to 30 S, small nuclear RNAs were detected which represented 6% of total RNA. The protein pattern was complex, and no clear-cut segregation of groups of proteins could be observed between the two fractions. They were both highly enriched in phosphoproteins as compared to nomoparticle proteins. In another fraction corresponding to the void volume of Bio-Gel chromatography, one-third of the RNA was small nuclear RNA. It is suggested that this fraction contains snRNP in addition to free proteins of molecular weight above 80 000 and to salt-resistant RNP similar to those described above but of small size. 相似文献
10.
R Berni V Lamberti 《Comparative biochemistry and physiology. B, Comparative biochemistry》1989,94(1):79-83
1. Retinol-binding protein is present in bovine serum essentially in the complexed form, bound to transthyretin, as in the case of the other mammalian retinol-binding proteins. 2. Bovine retinol-binding protein-transthyretin complex has the distinctive property of undergoing dissociation in the course of the DEAE-Sephadex chromatography of serum proteins. Therefore, the isolation of uncomplexed retinol-binding protein upon chromatography on this resin cannot represent evidence for lack of binding between it and TTR in bovine plasma, in contrast with previously reported conclusions (Heller J. (1975) J. biol. Chem. 250, (6549-6554). 3. Purified bovine retinol-binding protein and transthyretin can reconstitute a tight complex. 相似文献
11.
Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reverse-phase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins 相似文献
12.
Udby L Sørensen OE Pass J Johnsen AH Behrendt N Borregaard N Kjeldsen L 《Biochemistry》2004,43(40):12877-12886
Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein of unknown function and a member of the immunoglobulin superfamily. We demonstrate that CRISP-3 is a specific and high-affinity ligand of A1BG with a dissociation constant in the nanomolar range as evidenced by surface plasmon resonance. The A1BG-CRISP-3 complex is noncovalent with a 1:1 stoichiometry and is held together by strong electrostatic forces. Similar complexes have been described between toxins from snake venom and A1BG-like plasma proteins from opossum species. In these cases, complex formation inhibits the toxic effect of snake venom metalloproteinases or myotoxins and protects the animal from envenomation. We suggest that the A1BG-CRISP-3 complex displays a similar function in protecting the circulation from a potentially harmful effect of free CRISP-3. 相似文献
13.
The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions. 相似文献
14.
Dundr M Hebert MD Karpova TS Stanek D Xu H Shpargel KB Meier UT Neugebauer KM Matera AG Misteli T 《The Journal of cell biology》2004,164(6):831-842
Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures. 相似文献
15.
The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes 总被引:10,自引:0,他引:10
U Lindberg C E Schutt E Hellsten A C Tj?der T Hult 《Biochimica et biophysica acta》1988,967(3):391-400
In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin beta and profilin-actin gamma. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg2+, and can be crystallized. From the isolated profilin-actin complexes the beta- and gamma-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin. 相似文献
16.
C1 dissociation. Spontaneous generation in human serum of a trimer complex containing C1 inactivator, activated C1r, and zymogen C1s 总被引:1,自引:0,他引:1
A B Laurell U M?rtensson A G Sj?holm 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(12):4145-4151
Activation of the C1 complex in the presence of C1 inactivator (C1 IA) is known to result in the formation of tetramer C1 IA-C1r-C1s-C1 IA complexes that are dissociated from C1q. Both C1r and C1s of the tetramers are present in their activated forms. The present investigation concerned the generation of trimer complexes containing C1 IA, activated C1r, and zymogen C1s (C1 IA-C1r-C1s). C1 IA-C1r-C1s were released from C1q and were formed in high concentration during prolonged incubation (1 to 3 days) of normal serum at 37 degrees C without addition of activators. By contrast, dissociation of C1 with formation of C1 IA-C1r-C1s-C1 IA was complete within 30 min at 37 degrees C, when the serum was treated with heat-aggregated IgG (1 g/liter). On size exclusion chromatography (TSK-4000), C1 IA-C1r-C1s and C1 IA-C1r-C1s-C1 IA emerged with apparent m.w. of 320,000 and 460,000, respectively. The composition of the complexes was examined by absorption of serum with F(ab')2 anti-C1s- or anti-C1r-coated Sepharose beads. Eluates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting. Under nonreducing conditions, heat-aggregated IgG-treated serum showed high concentrations of C1 IA-C1r (m.w. 202,000) and C1 IA-C1s (m.w. 194,000), while serum incubated at 37 degrees C without activators showed high concentrations of C1 IA-C1r but no C1 IA-C1s. Under reducing conditions, heat-aggregated IgG-treated serum showed m.w. 120,000 and 110,000 complexes of C1 IA and the C1r and C1s light chains, respectively. Uncleaved C1s and the m.w. 120,000 complex was found in serum that was incubated at 37 degrees C without activators. Consistent with results obtained by size exclusion chromatography, analysis by crossed immunoelectrophoresis and by electroimmunoassay showed that C1s could be released from C1 IA-C1r-C1s in the presence of EDTA. 相似文献
17.
Gordon JM Shikov S Kuehner JN Liriano M Lee E Stafford W Poulsen MB Harrison C Moore C Bohm A 《Biochemistry》2011,50(47):10203-10214
Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1. 相似文献
18.
Saccharomyces cerevisiae cells harboring the gene encoding sarcotoxin IA secrete a peptide that is toxic to plant pathogenic bacteria. 总被引:4,自引:0,他引:4
R Aly D Granot Y Mahler-Slasky N Halpern D Nir E Galun 《Protein expression and purification》1999,16(1):120-124
Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly, Sarcophaga peregrina. Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced into Saccharomyces cerevisiae yeast cells and was expressed under a constitutive yeast promoter. The transformed yeast cells were grown in a liquid medium, and a peptide with a similar molecular size to that of the mature sarcotoxin IA was detected in the medium by Western blot analysis. The secreted sarcotoxin-like peptide (SLP) had a potent cytotoxic effect against several bacteria, including plant pathogenic bacteria, similar to the toxic effects of the authentic sarcotoxin IA. Erwinia carotovora was more susceptible to the toxic medium than Pseudomonas solanacearum and Pseudomonas syringae pv. lachrymans. Thus, yeast may be used in the production of such proteins for employment against various bacterial pathogens. 相似文献
19.
We have evaluated the effect of lysine guanidination in peptides and proteins on the dissociation of protonated ions in the gas phase. The dissociation of guanidinated model peptide ions compared to their unmodified forms showed behavior consistent with concepts of proton mobility as a major factor in determining favored fragmentation channels. Reduction of proton mobility associated with lysine guanidination was reflected by a relative increase in cleavages occurring C-terminal to aspartic acid residues as well as increases in small molecule losses. To evaluate the effect of guanidination on the dissociation behavior of whole protein ions, bovine ubiquitin was selected as a model. Essentially, all of the amide bond cleavages associated with the +10 charge state of fully guanidinated ubiquitin were observed to occur C-terminal to aspartic acid residues, unlike the dissociation behavior of the +10 ion of the unmodified protein, where competing cleavage N-terminal to proline and nonspecific amide bond cleavages were also observed. The +8 and lower charge states of the guanidinated protein showed prominent losses of small neutral molecules. This overall fragmentation behavior is consistent with current hypotheses regarding whole protein dissociation that consider proton mobility and intramolecular charge solvation as important factors in determining favored dissociation channels, and are also consistent with the fragmentation behaviors observed for the guanidinated model peptide ions. Further evaluation of the utility of condensed phase guanidination of whole proteins is necessary but the results described here confirm that guanidination can be an effective strategy for enhancing C-terminal aspartic acid cleavages. Gas phase dissociation exclusively at aspartic acid residues, especially for whole protein ions, could be useful in identifying and characterizing proteins via tandem mass spectrometry of whole protein ions. 相似文献
20.
Cytochrome c and plastocyanin form an electrostatic complex, which can be reinforced by amide bonds in the presence of a carbodiimide. Besides this cross-linking, carbodiimide also converts carboxylate side chains into neutral N-acylurea groups. Four derivatives of the covalent diprotein complex, which differ in the degree of this charge neutralization, are separated by cation-exchange chromatography. Electron-transfer reactions at different ionic strengths involving the electrostatic complex and the four derivatives of the covalent complex are studied by laser flash photolysis with flavin semiquinones as reducing agents. The reactivity of the associated proteins toward external reductants cannot be predicted simply on the basis of this reactivity of the separate proteins. Qualitative analysis of the dependence on ionic strength of the reactions between FMN semiquinone and the covalent derivatives indicates sites at which this reductant interacts with the cross-linked proteins. The surprisingly small steric shielding of the protein redox sites in the covalent complex, as deduced from the reactions at high ionic strength, may indicate that the proteins have multiple reaction domains on their surfaces or that the complex is dynamical or both. The intracomplex (unimolecular) electron-transfer reaction is fast in the electrostatic complex (ket = 1300 +/- 200 s-1) but undetectably slow in each of the four derivatives of the covalent complex (ket less than 0.2 s-1).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献