Methaemalbumin: a diagnostic surrogate for methaemoglobinaemia and treatment with red cell exchange in a patient with thalassaemia.

A man in his 30s with alpha thalassaemia (four-alpha globin gene deletion) presented with 1 week of shortness of breath and 1 month of general malaise. Pulse oximetry monitoring revealed low peripheral oxygen saturation of approximately 80% despite maximal high-flow nasal cannula oxygen (fractional inspired oxygen 1.0-60 L/min flow). Arterial blood gas samples were chocolate brown in colour, with a low arterial partial pressure of oxygen of 197 mm Hg. This large oxygen saturation gap raised suspicion for methaemoglobinaemia. However, the patient's co-oximetry results were suppressed by the blood gas analyser and delayed a definitive diagnosis. A methaemalbumin screen was sent instead, which was positive at 65 mg/L (reference interval: <3 mg/L). Treatment with methylene blue was initiated but did not result in complete resolution of cyanosis. This patient had been red cell exchange dependent since childhood for thalassaemia. Therefore, an urgent red cell exchange was initiated overnight, leading to an improvement in symptoms and interpretability of co-oximetry results. This resulted in rapid improvement without residual sequelae or complications. We conclude that a methaemalbumin screen can be used as a surrogate test for prompt confirmation of diagnosis in lieu of co-oximetry in cases of severe methaemoglobinaemia or in cases with underlying haemoglobinopathy. Red cell exchange can allow prompt methaemoglobinaemia reversal, especially if methylene blue is only partially effective.

Human Plasma and Recombinant Hemopexins: Heme Binding Revisited.

Plasma hemopexin (HPX) is the key antioxidant protein of the endogenous clearance pathway that limits the deleterious effects of heme released from hemoglobin and myoglobin (the term "heme" is used in this article to denote both the ferrous and ferric forms). During intra-vascular hemolysis, heme partitioning to protein and lipid increases as the plasma concentration of HPX declines. Therefore, the development of HPX as a replacement therapy during high heme stress could be a relevant intervention for hemolytic disorders. A logical approach to enhance HPX yield involves recombinant production strategies from human cell lines. The present study focuses on a biophysical assessment of heme binding to recombinant human HPX (rhHPX) produced in the Expi293FTM (HEK293) cell system. In this report, we examine rhHPX in comparison with plasma HPX using a systematic analysis of protein structural and functional characteristics related to heme binding. Analysis of rhHPX by UV/Vis absorption spectroscopy, circular dichroism (CD), size-exclusion chromatography (SEC)-HPLC, and catalase-like activity demonstrated a similarity to HPX fractionated from plasma. In particular, the titration of HPX apo-protein(s) with heme was performed for the first time using a wide range of heme concentrations to model HPX-heme interactions to approximate physiological conditions (from extremely low to more than two-fold heme molar excess over the protein). The CD titration data showed an induced bisignate CD Soret band pattern typical for plasma and rhHPX versions at low heme-to-protein molar ratios and demonstrated that further titration is dependent on the amount of protein-bound heme to the extent that the arising opposite CD couplet results in a complete inversion of the observed CD pattern. The data generated in this study suggest more than one binding site in both plasma and rhHPX. Furthermore, our study provides a useful analytical platform for the detailed characterization of HPX-heme interactions and potentially novel HPX fusion constructs.

Spectrophotometric evaluation of hemolysis in plasma by quantification of free oxyhemoglobin, methemoglobin, and methemalbumin in presence of bilirubin.

Severe intravascular hemolysis leads to the simultaneous presence of free heme pigments (oxyhemoglobin, methemoglobin, and methemalbumin) and bilirubin in human plasma. Standard spectrophotometric methods used to assess in vivo hemolysis inadequately address this complex analytical situation. Thus, we propose a novel quantification algorithm to ensure the highest analytical specificity. A corresponding second-derivative fitting algorithm was validated according to the guideline of bioanalytical method validation from the European Medicines Agency using plasma specimens (n = 1759) spiked with different concentrations of oxyhemoglobin and methemoglobin. The results were compared to standard spectrophotometric quantification methods described by Harboe, Noe, and Fairbanks. Based on the second-derivative method, simultaneous quantification of oxyhemoglobin and methemoglobin/methemalbumin in samples with total bilirubin concentrations ≤4.9 mg/dL (83.8 µmol/L) provided robust results (inaccuracy ≤20%, imprecision ≤16%). Analyzing UV/VIS spectra of plasma from patients with confirmed severe intravascular hemolysis evidenced an underestimation of up to 33% for the combined free heme pigment content. The employed second-derivative algorithm allows for automated and highly specific quantification of the free heme pigment content in diluted human plasma, which cannot be realized with standard spectrophotometric evaluation methods. An Excel-based tool readily applicable to clinical datasets accompanies this manuscript.

Heme Oxygenase Activity and Heme Binding in a Neonatal Mouse Model.

BACKGROUND: Severe hemolytic disease of the newborn leads to the release of pro-oxidative free heme (FH). Heme oxygenase (HO) is primarily responsible for detoxifying FH. OBJECTIVE: To investigate the protective effects of HO in a model of heme overload. METHODS: For in vitro studies, NIH3T3 HO-1-luc cells were incubated with 10, 30, or 60 µM FH or methemalbumin (MHA). HO-1 promoter activity was assessed 3, 6, and 24 h after treatment. Cell survival was indexed by viability assays. For in vivo studies, 1- and 5-week-old wild-type (Wt) or HO-1-heterozygous (Het, HO-1+/-) mice were given 60 µmol FH or MHA/kg intraperitoneally. After 24 h, plasma aspartate aminotransferease (AST)/alanine transaminase (ALT) and hemopexin, liver HO activity, and lipid peroxidation (LP) were determined. RESULTS: In HO-1-luc cells, HO-1 promoter activity peaked 6 h after incubation with 30 µM FH (1.6-fold) or 60 µM MHA (2.1-fold) over baseline. Twenty-four hours after exposure to 60 µM FH, a decrease in viability of 80% was found, compared with no decrease after exposure to 60 µM MHA. In 1-week-old Wt and HO-1 Het pups given 60 µmol FH/kg, HO activity significantly increased 3.5- and 3.1-fold, respectively. No changes in LP or AST/ALT levels were observed. In adult Wt and HO-1 Het mice, HO activity increased (3.0- and 2.6-fold, respectively). LP and AST levels significantly increased 28.4- and 2.7-fold, respectively, in adult HO-1 Het mice. Hemopexin levels at baseline were higher in adults compared with newborns for both Wt and Het mice. In addition, FH induced hemopexin levels in both adults and newborns, but to a lesser degree in newborns. CONCLUSIONS: FH is highly toxic in vitro, but its toxicity is abolished when bound to albumin. Newborns appear to be protected from the pro-oxidative effects of FH, which may be mediated by heme binding and a higher absolute HO activity at baseline and after FH-mediated induction.

Cola-colored dialysate as a gastrointestinal sequelae of cardiac surgery in a patient who underwent peritoneal dialysis.

The discoloration of effluent peritoneal dialysate, which is transparent in origin, is seen in some particular conditions including chyloperitoneum, calcium channel blocker usage, hemoperitoneum, perforated cholecystitis, iron administration, and hemorrhagic pancreatitis. We report a case of a 60-year-old woman who underwent peritoneal dialysis for 3 years and presented with conspicuous cola-colored (brownish-black) dialysate after a cardiac surgery. The findings of the dialysate analysis and the abdominal computed tomography showed that this discoloration could be due to the presence of methemalbumin caused by pancreatitis (not hemorrhagic) combined with intra-abdominal bleeding-both of which are rare gastrointestinal complications of cardiac surgery. She eventually died of pulseless electrical activity due to severe sepsis with profound shock. Therefore, the rare event of cola-colored peritoneal dialysate could present as severe gastrointestinal sequelae of cardiac surgery and may indicate a poor prognosis.

Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress.

Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.

[Antioxidant activity of oxygen-containing aromatic compounds].

Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H202-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20 degrees C in buffered physiological solution (pH 7.4) containing 6% DM F and 0.25% DMSO. The inhibitor's efficiency was quantitatively characterized by the inhibition constants (Ki, microM) or the inhibition degree (%). Ki values varied in the range of4 to 500 microM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds ofguaiacyl and syringal series exhibited high antioxidant activity (Ki in the range of 10-300 microM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycou maric acid.

Methaemalbumin formation in sickle cell disease: effect on oxidative protein modification and HO-1 induction.

Normally, cell free haemoglobin is bound by haptoglobin and efficiently cleared. However, the chronic haemolysis in sickle cell disease (SCD) overwhelms haptoglobin binding capacity and protein turnover, resulting in elevated cell free haemoglobin. Cell free haemoglobin acts as both a scavenger of vasoactive nitric oxide and a pro-oxidant. In addition, methaemoglobin (metHb) releases the haem moiety, which can bind to albumin to form methaemalbumin (metHSA). This study used electron paramagnetic resonance to detect metHSA in SCD plasma and demonstrated that haptoglobin prevents haem transfer from metHb to HSA. MetHSA may either provide a second line of defence against haemoglobin/haem-mediated oxidation or contribute to the pro-oxidant environment of SCD plasma. We demonstrated that HSA inhibited oxidative protein modification induced by metHb. Additionally, we showed that while metHb induced haem oxygenase 1 (HO-1), an indicator of oxidative stress, HSA attenuated metHb induction of this enzyme, thereby limiting the potential benefits of HO-1. Furthermore, HO-1 induction by metHSA was less than HO-1 induction by equimolar metHb not bound to albumin. Our findings confirm the presence of metHSA in SCD and suggest that haem transfer from metHb to HSA reduces the oxidative effects of free haemoglobin/haem on endothelium with both beneficial (reduced protein oxidation) and potentially harmful (reduced HO-1 induction) outcomes.

[Highly effective test-system for determination of the total antioxidant activity of human blood serum].

A highly effective test-system for quantitative characterization of the total antioxidant activity (TAA) of human blood serum (HBS), including methemalbumin (MetHa) as biocatalyst, H2O2 as the oxidant, o-phenylenediamine (PDA) as the acceptor of radicals and 2,2,5,7,8-pentamethylchroman-6-ol (PMC) as the inhibitor-calibrator, has been developed and proved under the laboratory environments. The test-system has been optimized for the concentrations of all the components, the reaction conditions and the PDA consumption monitoring at 37 degrees C in the medium of buffered physiological solution, pH 7.4 containing 5% DMFA and 0.5% DMSO. Under strongly standardized conditions by a comparison of the inhibiting action of the HBS and the calibrator PMC at 37 degrees C, the quantitative parameters of the HBS TAA were determined in microg of PMC, equivalent to 1 mg of HBS, or as a reverse value in mg of HBS, equivalent to the action of 1 microg of PMC. The values of the HBS TAA significantly varied for the group of healthy individuals and essentially decreased for the group of patients with thyroid gland pathologies. This underlies necessity of antioxidant therapy in these patients.

Purification and viral inactivation of hemoglobin from human placenta blood.

A new procedure was developed to obtain high-quality polymerized human hemoglobin by modifying purified hemoglobin with PLP and polymerized with GDA. Comparing polymerized hemoglobin products obtained from different methods, the product from the new procedure has similar physical, chemical, and biological properties in the molecular distribution, methemoglobin concentration, oxygen carrier capacity, P(50) and spectral analysis. Furthermore, the new procedure of modification after polymerization can save PLP greatly, and significantly reduce the cost. So the procedure of modification after polymerization is a better way in research and production.

Inhibition of heme oxygenase activity in newborn mice by azalanstat.

Inhibition of heme oxygenase (HO), the rate-limiting enzyme in heme catabolism, may be an ideal strategy for preventing neonatal jaundice. Although natural and synthetic heme analogs, called metalloporphyrins (Mps), have been extensively investigated for this purpose, some Mps are phototoxic, affect the activity of other enzymes, or induce HO-1 transcription-properties that may limit their clinical use. Another class of compounds, imidazole-dioxolanes, has been shown to selectively inhibit the inducible isozyme HO-1. Therefore, we investigated the efficacy of azalanstat (AZA), an imidazole-dioxolane, towards inhibiting HO activity in 7-day-old mice. We found that a single dose of AZA at 500 micromol.kg(-1) body mass (BM) administered i.p. significantly inhibited HO activity and reduced in vivo bilirubin production. In the spleen, HO inhibition (>50%) was observed within 0.25-3 h after administration. After 24 h, however, spleen HO activity, HO-1 protein, and HO-1 mRNA levels significantly increased 1.2-, 2.4-, and 4.0-fold, respectively. We conclude that AZA effectively inhibits in vivo HO activity only at a high dose and that it also induces spleen HO-1 gene transcription. Therefore, other imidazole-dioxolanes should be evaluated to determine whether they are more potent than AZA for use in treating neonatal jaundice.

[Substituted aminophenols and flavonoids as potential components for test-systems of total antioxidant activity].

A comparative kinetic study of ortho-phenylenediamine (PDA) oxidation in the "pseudoperoxidase" system Methemalbumin-H2O2 in the presence of 2-amino-4-tret-butylphenol (ATBP), 2-amino-4,6-di-tret-butylphenol (ADTBP) and its four N-acyl derivates, as well as flavonoids (quercetin, morin, silibin, hesperidin and naringin) has been carried out under standart conditions at 20 degrees C in phosphate buffered saline, pH 7.4, containing 5.25% ethanol and DMFA. ATBP, ADTBP and two its N-acyl-derivatives as well as all five flavonoids inhibited with different efficiency the PDA oxidation characterized in terms of the inhibition constants, K(i), M, or the percent of inhibition degree at the maximal taking concentrations of these inhibitors. Most effective antioxidants were quercetin (K(i) = 7.7x10(-5) M) and ATBP (K(i) = 1.26x10(-4) M). Using these characteristics and other necessary criteria, the pairs PDA-quercetin and PDA-ATBP were proposed for a practical application in the test-systems for total antioxidant activity of biological objects.

Effect of prototypic drugs ibuprofen and warfarin on global chaotropic unfolding of human serum heme-albumin: a fast-field-cycling 1H-NMR relaxometric study.

Human serum albumin (HSA) is the most prominent protein in plasma, but it is also found in tissues and secretions throughout the body. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme and drugs. HSA has been used as a model multidomain protein to investigate how interdomain interactions affect the global folding/unfolding process. Here, we report on the reversible chemical denaturation of heme-HSA involving three different conformational states (F, N, and B, occurring at pH 4.0, 7.0, and 9.0, respectively) and on the effect of prototypic drugs ibuprofen and warfarin on thermodynamics of the reversible unfolding process. Chaotropic unfolding of heme-HSA in the F, N, and B conformations is governed by different thermodynamic regimes, with the B form showing an entropic stabilization of the structure that compensates an enthalpic destabilization, and the F form easily unfolding under entropic control. Warfarin and ibuprofen binding stabilizes heme-HSA in both N and B states.

Binding of heme to human serum albumin: steady-state fluorescence, circular dichroism and optical difference spectroscopic studies.

The binding of monomeric heme to human serum albumin (HSA) was investigated using steady-state fluorescence, circular dichroism (CD) and optical difference spectroscopic (ODS) techniques. The existence of one strong binding site for heme on HSA was confirmed by titrating heme with HSA and following the quenching of tryptophan (Trp214) fluorescence emission intensity that occurred due to energy transfer. Up to around 1:1 stoichiometric ratio of HSA/heme, the quenching was observed to be very strong, however at higher ratios the quenching progressed very weakly. Similarly, the negative CD band centered at -397 nm, which appeared on adding heme to HSA, increased in intensity on sequential addition of heme up to [heme]/[HSA] = 1. Titration of HSA with heme was followed by ODS and the dissociation constant K(D) = (4.0 +/- 1.0) x 10(-5) M was deduced. Results have been explained on the basis of Michaelis-Menton type of mechanism for the heme binding, in which heme first binds reversibly to His146 at the surface of the protein to form an intermediate complex, followed by irreversible binding to Tyr161 in the interior of the protein.

The role of RBC destruction in vascular regions with high turbulence on atherosclerosis.

After intravascular red blood cell (RBC) destruction, released hemoglobin exceeding the binding capacities of haptoglobin and hemopexin would contribute, as free hemoglobin and/or hemin and/or methemalbumin, to the pathogenesis of atherosclerosis. Especially in some regions of vasculature with a high turbulence strong enough to destruct fragile or old RBCs, free hemoglobin concentrations can exceed locally the binding capacities of haptoglobin and hemopexin although their measured venous concentrations are normal. As a very simple model of this possible very local event we analysed free hemoglobin and methemalbumin levels after blood collection into evacuated tubes, and found that the increase of methemalbumin is more significant than free hemoglobin. The measurement of free hemoglobin and methemalbumin concentrations of arterial blood samples just from pre- and post-atherosclerotic lesions can likely shed new light on our understanding of the development of atherosclerosis.

Reductive activation of HCFC-123 by methaemalbumin.

Hydrochlorofluorocarbon 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), a close structural analogue of the hepatotoxic anaesthetic halotane and a replacement for some ozone-depleting chlorofluorocarbons, is metabolized by liver cytochrome P450 (P450), both in vitro and in vivo. P450 activates HCFC-123, both oxidatively and reductively, to reactive species which attack P450 itself and also damage other targets leading to hepatotoxicity. Previous work in our laboratory has shown that some haloalkanes, including halomethanes CCl4, CCl3Br, CHCl4 and CH2Cl2 as well as halothane, are activated by different haemoproteins to reactive metabolites resulting in the protein's suicidal inactivation. Among these is methaemalbumin (MHA), a synthetic complex of haem with human albumin often used as a model for various natural haemoproteins, such as P450. The aim of this study was to use MHA as a model to investigate the mechanism of P450 inactivation by HCFC-123. We found that MHA can reductively activate HCFC-123 to reactive species resulting in the loss of its haem group. During anaerobic incubation of MHA with 10 mM HCFC-123, a typical reduced difference spectrum was observed with a 470-nm peak that increased with time, indicating an interaction between HCFC-123 or HCFC-123 metabolites and haem. In similar anaerobic incubations, a significant loss of haem was measured using both the pyridine-haemochromogen technique and an ion-pairing reverse-phase HPLC method (37 and 30%, respectively). The loss of haem was time-, but not dose-dependent. No statistically significant loss of protoporphyrin IX, as measured by a fluorescence technique, or of the absolute haem spectrum produced in presence of CO (CO-haem complex) was observed up to 10 mM HCFC-123. Finally, a small but statistically significant inorganic fluoride production was measured in the presence of 20 mM HCFC-123 using an F(-)-specific electrode. Taken together, these results indicate that incubation of the non-enzymatic P450 model MHA with HCFC-123 under anaerobic conditions leads to reductive activation of the substrate, resulting in the modification of haem, as was previously shown to occur for halothane. The haem modification is due to interaction of the prosthetic haem group of MHA with HCFC-123 metabolites. These data confirm the results of previous work with rat liver microsomal P450 and confirm suicidal destruction of haem to be the mechanism responsible for the HCFC-123-dependent loss of the enzyme's content and catalytic function.

[Methemalbuminnemia after massive hemolysis during blackwater fever]. / Methémmalbuminémie après hémolyse massive au cours d'une fièvre bilieuse hémoglobinurique.

We report a case of blackwater fever with brown plasma due to the presence of methemalbumin. The discovery of plasma with this color is a rare event at the laboratory. This compound appears during intravascular hemolysis or hemorrhagic pancreatitis when the ability of haptoglobin and hemopexin to bind free hemoglobin has been exceeded. In these cases some of heme is oxidized to hematin and taken up by serum albumin to form an albumin-hematin complex called methemalbumin. The major clinical problem is to evoke the diagnosis of methemalbuminemia and not confuse with methemoglobinemia. In our case, methemalbumin was detected and quantified using a scanning spectrophotometer. Its diagnostic and clinicals consequences are discussed.

Effects of hypoxia on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells.

Although hypoxia induces heme oxygenase (HO)-1 protein and mRNA expression in many cell types, hypoxia has also been shown to decrease HO-1 mRNA and protein expression. We tested the hypothesis that 24-h preexposure to hypoxia in human placental preparations suppresses HO protein expression and enzymatic function. Immortalized HTR-8/SVneo first-trimester trophoblast cells and explants of normal human chorionic villi (CV) from term placentas were cultured for 24 h in 1%, 5%, or 20% O(2). HO protein levels were determined by Western blot analysis, and microsomal HO activity was measured. HO-2 protein content was decreased by 17% and 5% in human trophoblast cells after 24-h exposure to 1% and 5% O(2), respectively, versus 20% O(2). In contrast, HO-2 protein content in CV explants was unaffected by changes in oxygenation. HO-1 protein content, which was barely detectable in both biological systems, was not affected by changes in oxygenation. Similarly, HO enzymatic activity was unchanged in both preparations after 24-h exposure to 1%, 5%, or 20% O(2). The above data do not support the hypothesis that hypoxia in the human placenta suppresses both HO protein content and HO protein function. The present observations reinforce the necessity to determine both HO protein expression and function.

Spectroscopic studies on human serum albumin and methemalbumin: optical, steady-state, and picosecond time-resolved fluorescence studies, and kinetics of substrate oxidation by methemalbumin.

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity ( K(M)=433 microM, molar catalytic activity=0.33 s(-1)), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by approximately 80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (approximately 20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of approximately 3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the approximately 80% populated conformer of methemalbumin allows the orientation factor, kappa(2), to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 A was deduced.