Hemoglobin Beta Chain Research Articles

Preparation and physical characteristics of a hemoglobin solution modified by coupling to 2-nor-2-formylpyridoxal 5'-phosphate.

Stroma-free hemoglobin (Hb) solutions were prepared on a 20-I scale. In 3-I batches, the beta chains of hemoglobin were crosslinked with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP) that was synthesized on a gram scale. The coupling efficiency was 60 to 80 percent. The oxygen dissociation curve of these Hb/HbNFPLP mixtures was shifted to the right with P50 (PO2 for 50% saturation with oxygen) values of 26 to 38 torr versus values of 12 to 16 torr for the nonmodified hemoglobin solutions. Both the H+ Bohr factor and the Hill coefficient were lower for the Hb/HbNFPLP mixture than for the original hemoglobin solution. The oxygen-binding coefficient beta was the same for both types of Hb solutions. The viscosity and the colloid osmotic pressure of both solutions were also the same. During storage at 4 degrees C for 18 months, no precipitation or denaturation of hemoglobin was detectable in either solution. There was also no conversion of the modified hemoglobin molecules, HbNFPLP, to the native hemoglobin tetramers, dimers, or monomers. The percentage of methemoglobin remained at 5 percent for about 6 months; it increased to 26 percent over the next 12 months. The results indicate that intramolecular coupling of hemoglobin with NFPLP yields a stable product with physiologic oxygen-carrying properties.

Mapping of genes for cytochromes P-450b, P-450e, P-450g, and P-450h in the rat. Demonstration of two gene clusters with P450-b,e localized on chromosome 1.

Inbred ACI, WF, and RCS rats having characteristic markers for albino (c), hemoglobin beta-chain (Hbb), and pink-eyed dilution (p) on chromosome 1 and expressing variants for hepatic cytochromes P-450b, P-450e, P-450g, and P-450h were used in genetic mapping studies for these hemoproteins. The results of WF X (ACI X WF)F1 and RCS X (WF X RCS)F1 backcrosses revealed the existence of two gene clusters designated the P450-b,e and P450-g,h loci. The linkage map P450-b,e-p-c-Hbb on rat chromosome 1 was demonstrated and found to be congruent with Coh(P450-b,e)-p-c-Hbb on mouse chromosome 7. P450-g,h is not linked with P450-b,e and the other markers tested on rat chromosome 1. It appears that close genetic linkage, rather than common functional/regulatory properties, typify members of cytochrome P-450 families/subfamilies.

Linkage of albino and hemoglobin beta-chain loci in the rabbit.

Genetic linkage between the albino (c) and hemoglobin beta-chain (Hb beta) genes in rabbits was demonstrated by means of segregation data from one full-sib family. The map distance was estimated at 9 +/- 6 cM, which is similar to the 8 cM previously estimated between the same pair of loci in both mice and rats. The interspecies conservation of the chromosome segment comprising c and Hb beta thus extends outside the rodent family. Indications that it also may be conserved in man are discussed.

Homeothermic fish and hemoglobin: primary structure of the hemoglobin from bluefin tuna (Thunnus thynnus, Scromboidei).

Some fish are warm-bodied, e.g. the bluefin tuna (Thunnus thynnus), which has a muscle temperature 12-17 degrees C higher than its environment. This endothermy is achieved by aerobic metabolism and conserved by means of a heat-exchanger system. The hemoglobins of bluefin tuna are adapted to these conditions by their endothermic oxygenation, thus contributing to the preservation of the body energy. This is a new and so far unique property of tuna hemoglobin. The primary structure of the alpha and beta chains of bluefin tuna hemoglobins is presented. The sequence was determined after enzymatic and chemical cleavages of the chains and sequencing of the peptides in gas- and liquid-phase sequencers. The alpha chains consists of 143 residues and are N-terminally acetylated. The beta chains have 146 amino acids and show two ambiguities at positions 140 and 142. The alpha chains differ from the human alpha chains in 65 amino-acid residues, the beta chains in 76. The hemoglobins of bluefin tuna, carp and man are compared and their different physiological properties are discussed in relation to the sequence data. From the primary structure of tuna hemoglobins, it is possible to propose a molecular basis for their peculiar endothermic transition from the T to the R structure.

Post-synthetic modification in vivo of the major hemoglobin beta chain in the rat.

Rat hemoglobins are unusually heterogeneous among mammals. This heterogeneity provides multiple opportunities for asynchronies in synthesis and degradation. We have examined rat hemoglobin turnover after an intravenous injection of 2-14C-glycine. After analyzing incorporation into the seven nonallelic globin chains of adult rats, we found the percentage of the major beta chain decreases over the red cell lifespan while the percentage of one of the minor beta chains increases. This suggests that a post-synthetic modification event converts a portion of the latter into the former.

Interaction of hemoglobin and its component alpha and beta chains with band 3 protein.

The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity [Hill coefficient = 1.65 +/- 0.1; total number of binding sites at pH 6.6 in 5 mM sodium phosphate buffer = 32 500 +/- 940 pmol/mg; Ka = (3.0 +/- 0.5) X 10(5) M-1]. The binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis. The ratio of the interaction of hemoglobin to band 3 protein per se was 1:1, whereas the band 3 preparation as a whole (protein + lipids) was 3:1. Saturating levels of glyceraldehyde-3-phosphate dehydrogenase blocked only 33% of the total binding sites which were localized at the cytoplasmic segment; the remaining 67% was localized in lipids by their extraction with acetone. Reconstitution of acetone-extracted band 3 with phospholipid liposomes indicated phosphatidylserine as the binding site. The positive cooperativity in binding to acetone-extracted band 3 was increased (Hill constant = 2.1 +/- 0.1) compared to the band 3 preparation. After separation of the alpha and beta chains of hemoglobin, only the alpha chain binds to band 3 with positive cooperativity to an extent of 45-50% of native hemoglobin with similar affinity. The binding capacity of p-(hydroxymercuri)benzoate (HMB) derivatives of hemoglobin and its alpha chain was less than that of native hemoglobin, whereas HMB-beta chain or beta chain did not bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand binding to heme proteins: relevance of low-temperature data.

Binding of carbon monoxide to the beta chain of adult human hemoglobin has been studied by flash photolysis over the time range from about 100 ps to seconds and the temperature range from 40 to 300 K. Below about 180 K, binding occurs directly from the pocket (process I) and is nonexponential in time. Above about 180 K, some carbon monoxide molecules escape from the pocket into the protein matrix. Above about 240 K, escape into the solvent becomes measurable. Process I can be observed up to 300 K. The low-temperature data extrapolate smoothly to 300 K, proving that the results obtained below 180 K provide functionally relevant information. The experiments show again that the binding process even at physiological temperatures is regulated by the final binding step at the heme iron and that measurements at high temperatures are not sufficient to fully understand the association process.

Reaction of acetaldehyde with hemoglobin.

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the beta-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain.

A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.

Antigenic structure of human haemoglobin. Localization of the antigenic sites of the β-chain in three host species by synthetic overlapping peptides representing the entire chain

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.

Plasma protein alterations in AIDS and hemophilia A.

Plasma proteins of patients with AIDS, ARC, hemophilia A, and some viral infections were studied using various electrophoretic techniques, and compared to healthy control subjects. On isoelectric focusing (IEF) gels the most prominent and consistent finding was a marked increase in a basic protein band (AABP) at pI approximately equal to 9.0 in plasma samples derived from AIDS and hemophilia A patients. Using SDS gels, we noted an increased amount of protein in the 90 KD region in AIDS patients as compared to control subjects. On two dimensional gels (2D gels) basic protein(s) with pI congruent to 8.1-9.0 and molecular weight of approximately 90 KD were noted to be increased. In addition, a basic protein of 27 KD along with two acidic proteins in the low molecular weight region were also elevated in AIDS plasma. Although a limited number of samples were analyzed, it seems probable that a number of proteins are altered in AIDS plasma. AABP stained positive with periodic acid Schiff (PAS) reagent, indicating that it is a glycoprotein. This protein did not bind to an anti-IgG sepharose column, suggesting that it is not an immunoglobulin. The purified protein also did not react with antibody to fibrinogen and hemoglobin beta-chain. There are many changes in the plasma protein pattern of AIDS and hemophilia A patients as compared to normal controls.

Homology of kringle structures in urokinase and tissue-type plasminogen activator: the phylogeny with the related serine proteases.

Twelve amino acid sequences of kringle-forming polypeptides were compiled from the known sequences of urokinase A-chain (human), a tissue-type plasminogen activator (human), prothrombin (human and bovine), and plasminogen (human). Their sequence homologies with maximum match were examined by a computer program. A homology alignment and graphic matrix analyses did show that they had a great degree of homology. All the cysteine residues responsible for the kringle structures of urokinase and the tissue-type plasminogen activator were confidently preserved as well as other proteins. A phylogenetic tree was then reconstructed, and the A- and S-chain of bovine and human prothrombins were accounted for the measurement of the evolutionary time span. It was found that urokinase had a larger time span, as much as 60 million years (MY), than the tissue-type plasminogen activator. A common ancestral element of the kringle-related serine proteases was placed at around 500 MY ago, as old as the diversion of the alpha- and beta-chains of hemoglobin. Thus, the kringle-families have undergone a substantial evolutionary divergence. Moreover, they can be subgrouped into three subfamilies: plasminogen activators, plasminogen, and prothrombin A-chains, the last being the most distantly diverged prothrombin S-chains.

Monoclonal antibodies specific for sickle cell hemoglobin.

Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility.

Studies on the oxidation of hemoglobin Zurich (beta 63 E7 Arg).

Autoxidation and chemically-induced oxidation of hemoglobin Zurich (beta 63 E7 Arg) have been investigated by electron paramagnetic resonance and optical absorption spectroscopy. The results show that the replacement of the distal histidine of the hemoglobin beta chains by an arginine greatly enhances the susceptibility of the heme-iron to oxidative challenge. Both the kinetics and the products of the oxidation are pH dependent. Thus, at acidic and neutral pH, treatment of the protein with ferricyanide leads to a fast conversion of the oxy-protein to aquo-methemoglobin, which, eventually, is slowly converted to hemichromes. In contrast, the hydroxy-met derivative, formed upon chemical oxidation at high pH, is rapidly converted to hemichromes. The electron paramagnetic resonance features of the ferric derivatives of hemoglobin Zurich are somewhat singular, reflecting the modifications of the heme environment in the distal region of the abnormal chains. However, they can be related to heme complexes having their structural counterparts in oxidation products of hemoglobin A.

Carbon monoxide-driven reduction of ferric heme and heme proteins.

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.

Ligand binding kinetic studies on the hybrid hemoglobin alpha(carp):beta(human): a hemoglobin with a restricted allosteric range.

The preparation of native alpha- and beta-chains of carp hemoglobin and the preparation of the hybrid hemoglobin alpha(carp):beta(human), hybrid I, are described. CO association and dissociation kinetics were determined for the hybrid hemoglobin alpha(carp):beta(human) as a function of fractional saturation by stopped-flow and by flash and laser photolysis. Oxygen dissociation from the liganded distribution of conformers and overall oxygen association were studied by laser photolysis. An allosteric model was used to fit successfully all of the CO and oxygen kinetics and equilibria for this hybrid. In this model, it was assumed that the conformational changes were rapid with respect to ligand binding. A linear free-energy relationship relating rate constants and equilibrium constants was assumed in order to reduce the number of fitting parameters. In the allosteric model itself, it was assumed that L, but not c, varied with pH. For CO, the allosteric parameter c was 0.24; for oxygen, the value was 0.14. At pH 7, L was 80. In hybrid I, the calculated CO association rate constants for the R and T states differ by only a factor of 2. From the equilibrium data, with no assumptions as to the rate of conformational equilibrations , one can show that the allosteric model requires that the measured CO association constant from stopped-flow measurements be assigned to the T state. This state, however, is poised approximately midway between a carp-human T state and a carp hemoglobin R state. In this hybrid, neither chain shows normal R-state behavior; rather, the liganded state is also intermediate between R and T. In this hemoglobin, the low cooperativity appears to be associated with a very restricted range for the R-T conformational change.

Excitation transport and trapping in a synthetic chlorophyllide substituted hemoglobin: orientation of the chlorophyll S1 transition dipole.

Excitation transport in synthetic zinc chlorophyllide substituted hemoglobin has been observed by pico -second time-resolved fluorescence depolarization experiments. In this hybrid molecular system, two zinc chlorophyllide molecules are substituted into the beta-chains of hemoglobin, while deoxy hemes remain in the alpha-chains. The rate of excitation transfer between the two chlorophyllides is analyzed in terms of the distance and orientation dependences predicted by the F orster dipole-dipole theory. In this analysis, the beta-beta interchromophore geometry is assumed to be that of the deoxyhemoglobin crystal structure. When combined with steady-state fluorescence depolarization data of the complementary hybrid containing zinc chlorophyllide in the alpha-chains, these experiments provide the necessary information to determine the orientation of the S1 transition dipole moment in the zinc chlorophyllide molecule. We also find that the fluorescence lifetime of the zinc chlorophyllide is 1.42 ns when the heme is in the deoxy state but 3.75 ns when the heme is ligated to carbon monoxide. This is explained by irreversible excitation transfer from the S1 state of the zinc chlorophyllide to the lower energy excited states present in deoxy heme.

Involvement of His HC3 (146) beta in the Bohr effect of human hemoglobin. Studies of native and N-ethylmaleimide-treated hemoglobin A and hemoglobin Cowtown (beta 146 His replaced by Leu).

The involvement of the COOH-terminal histidines of the beta chains of human hemoglobin in the allosteric mechanism of oxygen binding has been the topic of intensive discussion. Data presented here on the functional properties of native and chemically modified forms of Hb Cowtown (beta 146 His replaced by Leu) suggest that approximately half of the alkaline Bohr effect is attributable to the imidazole of His HC3(146) beta. The contribution of this residue to the alkaline Bohr effect has been estimated variably as 40-60% and 15% or less. Equilibrium and kinetic studies show that the amino acid substitution in Hb Cowtown decreases the stability of the low affinity conformation, resulting in an increased oxygen affinity and altered sensitivity to anionic effectors. Detailed analysis of Hill plots of oxygen binding according to the Adair scheme reveals that, under conditions of moderate ionic strength (chloride = 0.1 M), the K2 and K3 values for Hb A and Hb Cowtown differ, whereas the K1 and K4 values are closely similar over the physiological pH range. The decreased pH sensitivity of Hb Cowtown is associated with a decreased pH sensitivity of K1, the first Adair constant. In contrast to des-His(146 beta) hemoglobin, the cooperative interactions shown by Hb Cowtown under conditions of moderate ionic strength are not reduced in comparison to those of Hb A. This and the similarity of K1 and K4 values for Hb A and Hb Cowtown indicate that under these conditions the salt bridge formed by the COOH-terminal imidazole group does not significantly contribute to the free energy difference between "T-state" and "R-state" hemoglobin. It appears that the salt bridge formed by the COOH-terminal carboxyl group stabilizes the deoxy, T-state, conformation to a greater degree than previously appreciated. Chemical modification of the Cys(93 beta) residue of Hb Cowtown with N-ethylmaleimide causes a decrease in its oxygen affinity, in contrast to the increase in affinity exhibited by N-ethylmaleimide-modified Hb A. Hemoglobins A and Cowtown have remarkably similar oxygen binding properties after this modification and are shown to have K1 and K4 values distinctly different from those of unmodified Hb A. The properties of native and chemically modified forms of Hb Cowtown are indicative of a large contribution of the His HC3 (146) beta residue to the alkaline Bohr effect and also illustrate how chemical modifications or changes of strategic amino acid residues can result in pronounced differences in the conformational equilibrium of an allosteric protein.

Reductive hydroxyethylation of hemoglobin A. Functional properties of hemoglobin A selectively hydroxyethylated or dihydroxypropylated at the alpha-amino groups.

The reactivity of the amino groups of hemoglobin A toward reductive hydroxyethylation with glycolaldehyde in the presence of NaCNBH3 has been investigated. The alpha-amino groups of the alpha- and beta-chains of hemoglobin A are the most reactive amino groups of the protein for reductive hydroxyethylation; both sites are about equally reactive. Similarly, on reaction of hemoglobin A with glyceraldehyde in the presence of NaCNBH3, the alpha-amino groups of both the alpha- and beta-chains of the protein undergo selective reductive dihydroxypropylation. Modification of the alpha-amino groups of hemoglobin A either by reductive hydroxyethylation or reductive dihydroxypropylation apparently lowers the pKa of the alpha-amino groups and results in an early elution of the modified protein from CM52 chromatography compared with that of the unmodified protein as well as protein modified at the epsilon-amino groups. By hybridization of the modified chains with unmodified chains, new derivatives of hemoglobin with dihydroxypropyl or hydroxyethyl moieties on Val-1 (alpha) or Val-1(beta) of the protein have been prepared for the functional studies. Modification of the alpha-amino group of Val-1(alpha) by reductive hydroxyethylation or by reductive dihydroxypropylation resulted in an increased oxygen affinity of hemoglobin A, whereas the same modification of the alpha-amino group of the beta-chain resulted in a slight decrease in the oxygen affinity. The Hill coefficient of all four derivatives was 2.7, nearly the same as that of the native protein, showing normal heme-heme interactions in these derivatives. Both 2,3-diphosphoglycerate and chloride modulate the oxygen affinity of these hybrids; the influence of 2,3-diphosphoglycerate on decreasing the oxygen affinity of the hybrids with alkyl groups at Val-1(beta) is relatively less than that of the native molecule. On the other hand, when the modification was on Val-1(alpha), the relative decrease in oxygen affinity of the hybrids upon binding of 2,3-diphosphoglycerate was of the same order as with the native protein. The chloride ion-mediated relative decrease in the oxygen affinity of the hybrids with alkyl groups at Val-1(beta) was nearly the same as that seen with the Val-1(alpha) hybrids, and lower than that with the native molecule. The relative influence of hydroxyethylation or of dihydroxypropylation at a given site was nearly the same, suggesting that the size of the alkyl chain has little to do with the observed changes in the oxygen affinity. The influence of reductive alkylation of the alpha-amino groups with these two hydroxyaldehydes probably reflects the change (decrease) in the pKa of the reductively hydroxyethylated or dihydroxypropylated alpha-amino group as compared with that of the unmodified alpha-amino groups.

Nanosecond flash photolysis study of carbon monoxide binding to the beta chain of hemoglobin Zürich [beta 63(E7)His leads to Arg

Binding of carbon monoxide to beta chains of hemoglobin Zürich has been studied by flash photolysis over the time range of nanoseconds to seconds at temperatures from 20 to 300 K. From 20 to 200 K a single rebinding process (process I) is seen, characterized by a distribution of barrier heights with a peak enthalpy of 2.3 kJ/mol. Above 200 K some ligands escape from the pocket into the matrix, and above 260 K recombination from the solvent sets in. Process I is visible up to 300 K, but above 200 K its rate remains essentially constant at about 4 X 10(8)s -1. Above about 250 K, process I is exponential in time, indicating rapid conformational relaxation. The results are discussed within the framework of a sequential model for ligand binding.