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Multicomponent Analysis of Heme Protein Spectra in Biological Materials
ANALYTICAL BIOCHEMISTRY
233, 115–123 (1996)
Article No. 0015
Multicomponent Analysis of Heme Protein Spectra in Biological Materials James A. North, Dietrich Rein, and Al L. Tappel Department of Food Science and Technology, University of California, Davis, California 95616
Received August 1, 1995
Absorption spectra of heme proteins in a tissue homogenate contain information about the oxidation status of the biological sample. Deconvolution of absorption spectra can be used to quantitate the amount of individual heme proteins present in the mixture. The heme spectral analysis program (HSAP), a computer spreadsheet program, was used to quantitatively calculate values for heme proteins measured spectrally (390 to 450 and 500 to 640 nm) in tissue homogenates undergoing oxidation. The amount of oxidized heme proteins obtained by HSAP can be compared to other measurements of tissue oxidation. Precise quantitation of the amount of heme proteins present in a homogenate sample provided accurate assessment of the oxidized heme proteins calculated by HSAP. This quantitation was achieved through modification of existing pyridine hemochrome methods. Input into HSAP of the total heme protein content via the pyridine hemochrome value generated reproducible values for oxidized heme proteins. The program has broad potential as a multicomponent analysis tool. Modification of HSAP led to the development of a difference spectra analysis program (DSAP) which was used to quantitate the type and amount of heme proteins observed in mitochondrial difference spectra. In the present application, HSAP and DSAP provide methods for interpreting complex spectral information of multicomponent biological samples that undergo oxidation. q 1996 Academic Press, Inc.
In biological systems some oxidative events are beneficial, such as the detoxification of xenobiotics, whereas others are disastrous, e.g., the uncontrolled peroxidation of membrane lipids. Investigation into mechanistic details of oxidative processes in biological samples often requires quantitation of the damage observed in proteins, lipids, and other cellular components. Damage caused by oxidative reactions can be measured by a variety of assays that analyze the generation of prod-
ucts formed during the oxidative event. Of the many products generated, the oxidation of heme proteins represents a unique class of compounds that can be used as markers in the oxidative process. Heme proteins contain an iron–porphyrin complex capable of accepting unpaired electrons from various sources. Oxidation of the iron affects the energy state of the porphyrin ring and ultimately results in distinctly different spectra of the reduced and oxidized states. Spectral scans that read absorbance values across a range of visible wavelengths (390 to 640 nm) contain information about the oxidation state of individual heme proteins such as hemoglobin, myoglobin, or mitochondrial and microsomal cytochromes. Information derived from complex heme spectral scans obtained for samples originating from tissues, homogenates, or subcellular fractions can be obtained through comparison to reference spectra by multicomponent analysis. Although methods using multicomponent analysis are employed in laboratories to analyze the concentration of components from spectral data, the standard practice is to select specific wavelengths and, knowing the extinction coefficients of the different compounds at those wavelengths, solve the simultaneous equations generated. Existing methods of quantitation mostly measure irreversible oxidative products. A heme protein spectral analysis program previously developed in this laboratory is a method of deconvoluting composite spectra (500 to 640 nm) and obtaining molar ratio values of heme proteins in tissue samples (1). This heme protein spectral analysis program, measuring reversible and irreversible damage to heme proteins, uses a spreadsheet program that analyzes absorbancies at many points in the spectrum rather than a few wavelengths and a combination of regression analysis and successive approximations. Tissue homogenate samples contain a high amount of turbidity. This can be reduced by subtracting an appropriate level of light scatter from a background scan prior to acquisition of spectral data. 115
0003-2697/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Several layers of parafilm have been found to be adequate under most circumstances to simulate some of the background turbidity. Light scatter is not linear with respect to wavelength; therefore, a turbidity calculation had been introduced to the heme protein spectral analysis program (1, 2). In the present study, absorbance values corresponding to the gamma peaks of the various heme proteins (390 to 450 nm) have been added to the previous program and form a major contribution to the new heme spectra analysis program (HSAP).1 With the previous program (1) we could adequately analyze reduced hemoglobin species. However, measurement of oxidized heme proteins presented a unique problem in unambiguously assigning individual spectra. Oxidized heme proteins of the mitochondria, microsomes, the oxidized, partially denatured hemoglobin compound hemichrome, and methemoglobin produce spectra that closely resemble each other in the region from 500 to 640 nm. One solution was to include spectral data in HSAP for these oxidized heme proteins arising from the strongly absorbing Soret region containing the g peaks. In this region, peak maxima for oxidized heme components are sufficiently different for discrimination with HSAP (methemoglobin, 406 nm; hemichrome, 416 nm; combined oxidized mitochondria including cytochrome aa3, cytochrome b, and cytochrome c, 414 nm; and combined oxidized microsomes including cytochrome P450 and cytochrome b5, 425 nm). Thus, the recording of g peaks allows additional assignment of oxidized heme proteins originating from various sources, including mitochondrial or microsomal cytochromes that are normally hidden in spectra because of the overwhelming amount of hemoglobin present in nonperfused tissue homogenates. HSAP was also constrained to solve spectra obtained through oxidation of heme proteins by recognizing chemical reactions that can occur in the samples. For example, fully oxygenated homogenate samples should not contain reduced hemoglobin but instead oxyhemoglobin, so the optimization portion of the HSAP did not include deoxygenated, reduced hemoglobin in its calculations. Further refinements of HSAP based on the chemical reactions possible in the homogenate can be used when solving multicomponent spectra. These refinements may include fixing the amount of mitochondria and microsomes present in a given sample. Values for the amounts of organelles in a particular tissue homogenate can be estimated by reviewing the literature (3–5). A further aid in analyzing the spectra to be deconvoluted and quantitated by the modified HSAP is the 1 Abbreviations used: HSAP, heme spectra analysis program; PyrHo, pyridine hemochrome; DSAP, difference spectra analysis program.
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use of the pyridine hemochrome (PyrHo) technique to quantify the total amount of heme proteins in highly turbid homogenate samples (6, 7). Routine hemoglobin measurements often utilize cyanide solutions to convert hemoglobin to the cyanomethemoglobin derivative (8). Turbidity present in tissue homogenates interferes with measurements of cyanomethemoglobin. PyrHo, however, is measured by difference spectra so that turbidity does not contribute to the final calculations. Therefore, PyrHo values were used in HSAP as an independent fixed parameter. This PyrHo method has two advantages, the minimization of interference due to turbidity in tissue homogenates and the vivid illustration of the large amount of hemoglobin present in samples that can obscure spectral absorbance from cytochromes and other heme proteins. To aid in the deconvolution of the adjoining a and b peaks of cytochromes of organelles, the difference spectra analysis program (DSAP) was developed in the present investigation. Difference spectra are routinely used to identify the heme components of organelles such as mitochondria. Spectral values of oxidized cytochrome standards were subtracted from the spectral values of reduced cytochrome standards to provide a reference for this multicomponent difference analysis. The dominant absorbance peaks, or a peaks, observed in difference spectra represent absorbance changes for cytochrome aa3 (605 nm), cytochrome c (550 nm), and cytochrome b (560 nm, usually as a shoulder of the cytochrome c peak). The b peaks for these components appear near 518 nm; the soret bands appear in the 430 to 450 nm region. Although enough information is available to provide evidence for the type of heme protein present in a mitochondrial difference spectra, quantitation of the individual components by using the entire difference spectra routinely has not been done. This multicomponent analytical technique facilitates the quantitation of individual heme proteins present in a mitochondrial sample. This paper demonstrates the use of HSAP and its sister program DSAP to measure changes in the oxidative state of heme proteins in tissues and other biological systems. The programs can be relevant to some aspects of clinical problems associated with tissue injury that involve free radical reactions. MATERIALS AND METHODS
Chemicals. Rat and human hemoglobins and sodium dithionite were purchased from Sigma Chemical Co. (St. Louis, MO); pyridine and other solvents were from Fisher Scientific (Pittsburgh, PA). Animals. Tissue homogenates were prepared from male Sprague–Dawley rats fed synthetic diets as described previously (2, 9). Tissue homogenates in Krebs–Ringer phosphate buffer (10% w/v) were trans-
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ferred to glass serum bottles and closed with rubber stoppers. The homogenates were incubated in a Gyrotory water bath shaker (New Brunswick Scientific Co., Inc., New Brunswick, NJ) at 377C with continuous shaking (180 cycles/min). Spectrophotometric measurement of heme proteins from tissue homogenates. The absorbance spectra of tissue homogenates were obtained with a Beckman DU-50 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). Tissue homogenates were transferred to a cuvette with a light path of 10 mm containing equal volume buffer (final tissue concentration: 50 mg/ ml) and mixed. Parafilm, representing turbidity, was used as a background to subtract some of the absorbance caused by turbidity inherent in tissue homogenates. Samples were scanned from 390 to 450 nm (gregion) at a 101 dilution and from 500 to 640 nm (a and b regions). Absorbance vs wavelength at 2-nm intervals was automatically recorded by a scan program in the spectrophotometer. Spectral data obtained for fresh homogenates and for incubant samples were transferred to the spreadsheet computer program. Analysis of absorbance spectra of heme proteins of tissue homogenates. HSAP is a spreadsheet program written with Excel 5.0 (Microsoft Corp., Redmond, WA) that contains the micromolar absorbance values from 390 to 450 nm and from 500 to 640 nm of the visible spectra of individual standard heme proteins. The analysis is based on the knowledge that the absorbance spectrum of a mixture of heme proteins closely obeys Beer’s law and is the sum of the spectra of the individual heme proteins including any contribution from turbidity. Quantitation is achieved by matching a spectrum calculated from a combination of standard spectra with the experimental spectrum through successive approximations. These calculations can be automated through the use of the spreadsheet program solver or optimization function (Frontline Systems, Inc., Incline Village, NV). Development and applications of multicomponent analysis programs have been previously described (10–13). Specific details about a heme protein spectra analysis program can be obtained from Chen and Tappel (13). Modifications described in the present study are: (1) 2-nm intervals from 390 to 450 and from 500 to 640 were entered into HSAP; (2) turbidity contribution to the spectrum absorbance was calculated by using the light scattering formula: Turbidity Å alb, where a and b represent constants specific to that particular homogenate mixture at the wavelength l; and (3) total heme proteins, measured by the pyridine method, were added as a constraint for HSAP. Total heme quantification by the pyridine hemochrome method. Conversion of heme proteins to their PyrHo derivative was used to determine the total amount of heme proteins present in the tissue homoge-
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FIG. 1. Pyridine hemochrome standard curve generated using cyanomethemoglobin as a standard reference. The concentration per gram rat hemoglobin determined by the cyanomethemoglobin method was plotted against the absorbance difference per gram rat hemoglobin obtained by the PyrHo technique and is shown as data points (j). The regression line drawn through the points has a slope Å 53.8 mM heme per unit of absorbance difference, R 2 Å 0.98.
nates (14). Briefly, an equal volume of a stock solution containing 200 mM NaOH, 40% vol pyridine and 0.8 mM K3Fe(CN)6 was added to an aliquot of the tissue homogenate and vortexed. This oxidized sample was used to blank the spectrophotometer for the subsequent scan of the reduced sample. Sodium dithionite at a final concentration of 0.02 mM was added to this mixture to reduce hemichromes to hemochromes and after 3 min the maximum and minimum absorbancies were obtained from the interval between 535 to 560 nm. The minimum absorbance around 539 nm (a – b valley) was subtracted from the maximum absorbance around 555 nm (a peak). An extinction coefficient of 19.2 mM01 for PyrHo (see calibration below) was used to calculate the molar tissue heme concentration for each sample. The molar extinction coefficient for PyrHo was obtained by combining and calibrating two independent methods used to quantify total heme components. Literature values for the molar extinction coefficient of the PyrHo spectrum vary greatly with a range between 19 and 24 mM01. We calibrated the PyrHo assay for tissue homogenates against the generally accepted cyanomethemoglobin technique for quantitation of heme compounds (3, 15) using a rat hemoglobin standard (Fig. 1; R 2 Å 0.98). Subjecting the hemoglobin standard solution to PyrHo analysis as outlined above resulted in an extinction coefficient of 19.2 mM01. Both the cyanomethemoglobin and PyrHo methods produced similar results when a low turbidity standard was used. The PyrHo method, however, is far less susceptible to interference by trubidity since it is based on difference spectra analysis. It was therefore used to quantify total heme compounds in the homogenate samples.
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Analysis of difference spectra of heme proteins of mitochondria. DSAP is a spreadsheet program written with Excel 4.0 (Microsoft Corp.) and contains millimolar absorptivities from 500 to 640 nm of the visible spectra of the major heme containing proteins present in mammalian mitochondria, cytochromes aa3 , cytochrome b, and cytochrome c. Adding other heme protein spectra, e.g., cytochrome c1 , would not alter the analysis greatly because the additional spectra would fit well within one of the three major species included in the DSAP. Although the maximum and minimum absorbancies may be different for different cytochromes within the same family, we concluded that the various difference spectra obtained by subtracting the absorptivity values of the oxidized form of the cytochrome from the reduced form were similar. The theory for analysis and quantitation is similar to that for HSAP except that values are obtained by successive approximations of difference spectra in which the oxidized spectral absorbancies have been subtracted from the reduced spectral absorbancies of heme proteins. Cytochrome millimolar absorptivities were calculated from spectra obtained from the literature (3–5, 16–21). Spectra from the literature were scanned and digitized using software available from the NIH (Image 1.49, NIH, 1989). Statistical evaluation. Data are expressed as means { SE. All observations were performed with at least triplicate samples. RESULTS
Application of HSAP to Heme Protein Spectra Figure 2A shows spectral changes of heme-containing proteins during oxidative reactions. Before incubation, the dominant absorbance peaks appearing around 530 and 575 nm indicate that oxyhemoglobin is the major component. The location of the soret band at around 415 nm supports this finding. After 2 h incubation, spectral changes occurred in both regions showing the formation of methemoglobin and other oxidized heme proteins. Standard spectra of oxidized heme proteins are shown in Fig. 2B. HSAP Analysis of Published Spectra To ascertain the reproducibility and accuracy of HSAP with respect to the identification and tentative quantitation of hemoglobin species, published spectra were subjected to multicomponent analysis. The results are shown in Fig. 3. Figure 3A shows absorbance values from a spectrum of human hemoglobin (22) reduced by addition of sodium dithionite and the model spectrum calculated with HSAP based on its determination of heme protein concentrations. HSAP calculated the concentration of hemoglobin to
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FIG. 2. Spectral changes of heme proteins. (A) The changes in liver tissue homogenates incubated at 377C. Soret peaks are 1/10 original values (380 to 450 nm). Top spectra, 0 h; bottom spectra, 2 h. (s) Spectral absorbance data points; (—) spectra calculated with HSAP where, in the 500 to 640 nm region, it is obscured by the absorbance data points. (—) Lines under spectra show turbidity calculated with HSAP. (B) The individual and composite standard spectra that can contribute to the oxidized spectra observed after 2 h incubation. The lines represent (—) methemoglobin, (---) hemichrome, and (– –) oxidized heme proteins of mitochondria.
be approximately 2.7 mM compared to the published value of 3.0 mM. HSAP analyses were performed independently of the values found in the literature, thus the agreements on concentrations show the usefulness of this program. Kindt et al. (22) also measured methemoglobin, the oxidized form of hemoglobin, and the absorbance values are shown in Fig. 3B along with the model spectrum calculated with HSAP. On the basis of its determination of heme protein concentrations, HSAP calculated the concentration of methemoglobin to be approximately 3.03 mM compared to a published value of 3.0 mM. Figure 3C shows absorbance values from a spectrum of oxygenated sperm whale myoglobin (23) and the model spectrum calculated with HSAP based on the determination of heme protein concentrations. Myoglobin and metmyoglobin spectra resemble hemoglobin and methemoglobin spectra such that HSAP calculated the concentration to be approximately 9.9 mM compared to the published value of 10 mM. Shikama and
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chromes of the mitochondria as the heme proteins responsible for the spectral data in the proportions expected in rat heart (26), approximately 38 and 62%, respectively. The results shown in Fig. 3 demonstrate the ability of HSAP to supply good calculated fits to the experimental data. HSAP approximations also support the published qualitative description of the spectra and is in accord with the expected biochemistry. The published spectra used contain only one or two hemoglobin species whereas tissue and homogenate samples represent complex spectra containing multiple heme protein mixtures. Heme Quantification in Tissues
FIG. 3. Analysis of absorbance spectra of heme proteins from the literature with HSAP. Description of analysis is under Materials and Methods. Sources are cited under Results. (l) Values determined from spectra from the literature; (—) model spectra calculated with HSAP. The (---) line under the spectra is the absorbance calculated for turbidity with HSAP.
Matsuoka (23) also measured metmyoglobin, the oxidized form of myoglobin, and both the absorbance values and the calculated spectra are shown in Fig. 3D. Via HSAP, the metmyoglobin concentration was calculated to be approximately 9.3 mM compared to the published value of 10 mM. Figure 3E shows molar absorptivity values from a spectrum of oxygenated lugworm hemoglobin (24) and the spectrum calculated with HSAP. Since the millimolar absorptivities were used as the experimental absorbance values to calculate the millimolar concentrations of heme proteins in HSAP, the expected concentration of oxygenated hemoglobin should be approximately 1000 mM. HSAP calculated a concentration of HbO2 in the lugworm to be 961 mM, in good agreement with the expected value. Figure 3F shows absorbance values from a spectrum of isolated adult rat heart cells under anaerobic conditions (25), and the simulated spectrum calculated with HSAP. Because the published spectrum provided only relative measurement of absorbance, HSAP could only be used to provide qualitative information. In spite of these limitations, HSAP matched the experimental spectrum using only deoxymyoglobin and reduced cyto-
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Measuring the amount of heme protein in blood samples routinely consists of converting all forms of the heme into a common spectrally defined derivative, such as cyanomethemoglobin (8, 15). The turbidity present in tissue homogenates, however, directly interferes with the measurements of cyanomethemoglobin. Similar to the cyanomethemoglobin method, the pyridine hemochrome technique does not depend on the initial ligand state of hemoglobin to produce a spectrally defined common derivative (6). Turbidity, however, does not interfere with PyrHo measurements. The extinction coefficient of 19.2 mM01cm01 was used to convert PyrHo values to heme protein molar concentrations for determining the amount of heme compounds in a biological system. Addition of an alkaline solution containing pyridine and the reducing agent sodium dithionite stoichiometrically converts heme iron porphyrins to hemochromes in the heme mixtures tested. The hemochrome structure has a sharp absorption maximum around 556 nm whereas the corresponding oxidized porphyrin–pyridine adduct, hemichrome, has a diffuse spectrum with maxima near 534 and 556 nm. The absorption difference between reduced and oxidized PyrHo minimizes spectral interference due to turbidity and thus can be used to quantify the amount of PyrHo present in the heme protein samples (7). The calibration of PyrHo using rat hemoglobin or tissue homogenates resulted in a linear relationship between the amount of heme calculated to be present in the solution and the difference in absorbance between reduced and oxidized pyridine adducts (Fig. 1, rat Hb; R 2 Å 0.98) (Fig. 4, rat liver homogenate; R 2 Å 0.99). The results obtained with liver homogenates prepared from basal diet-fed animals showed that the PyrHo method produced consistent heme protein values (Table 1). When the liver homogenate at a sample concentration of 0.05 g/ml was assayed in several different preparations, the values were consistent within groups as well as between groups. When the liver homogenate concentration was varied, the total heme pro-
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NORTH, REIN, AND TAPPEL TABLE 2
Pyridine Hemochrome Replication
FIG. 4. Heme content of liver samples as a function of homogenate concentration. Total heme concentration was determined by the pyridine hemochrome method outlined under Materials and Methods. (j) Data points represent results from three different rat livers. The regression line has a slope of 0.372 mM heme/g liver; R 2 Å 0.99.
tein content calculated by the PyrHo technique was similar; 0.29 { 0.01 mmol heme protein/kg liver (Table 2, average { SE). These results indicate that PyrHo can provide reproducible quantitation of the total amount of heme proteins in tissue homogenates. Another assay used to support the accuracy of the PyrHo method is a recovery experiment. Hamster red blood cells were assayed for total heme concentration in the presence or absence of a known amount of rat hemoglobin standard. Even when the concentration of the red blood cell was varied, all of the hemoglobin standard could be accounted for in the mixture (Table 3). Although the PyrHo method gives reproducible results, the total amount of heme proteins will vary from tissue to tissue. This variance depends mainly upon the amount of perfusion and if the organ were subjected to prior storage and experimentation. However, since the total amount of heme proteins is small in comparison to the total amount of proteins in the entire sample,
TABLE 1
Pyridine Hemochrome Replication
Tissue concentration (mg/ml)
Total heme value (mmol/kg tissue)
50 50 25 25 12.5 12.5
0.30 0.28 0.24 0.29 0.29 0.30
Average SE
0.29 0.01
hemoglobin can serve as a marker for oxidation independent of its actual concentration. In fact, when tissue homogenates containing a wide divergence in the amounts of hemoglobin were allowed to oxidize for 2 h, the percentages of oxidized heme proteins measured by HSAP were not significantly different (duplicate samples of rat heart homogenates containing 19, 21, and 91 mM PyrHo produced 82, 80, and 86% oxidized heme protein values, respectively). The absolute amount of oxidized heme proteins differed between the various preparations (16, 17, and 78 mM oxidized heme proteins, respectively). Thus, comparisons between different preparations of the same tissue, the same tissue from different animals, or between different tissues can effectively be normalized and subjectively interpreted (for examples, see Refs. 1, 10, 11, 27, 28). Application of DSAP to Mitochondrial Difference Spectra Figure 5 represents difference spectra obtained from the literature and analyses of the spectra calculated with DSAP. The calculated spectra confirm the experimental data in each case. Turbidity does not interfere in these spectra because difference spectra are used to cancel out turbidity. Another advantage of the DSAP is the emphasis of the cytochrome b peak contribution
mmol heme/kg tissue TABLE 3 Group:Sample No.
Total heme value
Group average { SE
A:1 A:2 A:3 A:4 B:1 B:2 B:3 C:1 C:2 C:3
0.24 0.24 0.23 0.32 0.28 0.29 0.30 0.26 0.28 0.34
0.27 { 0.02
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Recovery of Heme Protein Total heme value (mmol/kg tissue)
Hemoglobin sample 0.29 { 0.01
0.29 { 0.02
Red blood cell Hemoglobin standard Red blood cell plus hemoglobin standard Recovery
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0.1% red blood cell by volume
0.2% red blood cell by volume
24 { 1.2 64
49 { 2.4 64
91 { 0.4
114 { 1.1
104%
101%
MULTICOMPONENT ANALYSIS OF HEME SPECTRA
FIG. 5. Analysis of difference spectra of mitochondrial heme proteins from the literature with DSAP. Description of analysis is under Materials and Methods. Sources are cited under Results. (l) Values determined from spectra from the literature; (—) model spectra calculated with DSAP.
to the overall spectrum. Even in the presence of large amounts of cytochrome c, DSAP reproducibly assigned values for cytochrome b in samples that contained both cytochrome c and cytochrome b. Figure 5A shows a difference spectrum of pigeon heart mitochondria (29) and the model difference spectrum calculated with DSAP based on its determination of cytochrome concentrations. Using DSAP, relative concentrations were calculated for cytochrome aa3 , cytochrome b, and cytochrome c at 52, 4, and 44%, respectively, of the total cytochrome content. Despite the absence of compositional data, the calculated spectrum was in accord with the experimental spectrum. Figure 5B shows a difference spectrum of crab flexor muscle (30) and the simulated spectrum calculated with DSAP. The percentage compositions of cytochrome aa3 , cytochrome b, and cytochrome c were calculated via DSAP to be 62, 23, and 15%, respectively, compared to the published values of 72, 7, and 21%, respectively. Figure 5C shows optical density changes in the difference spectrum of anoxic toad sartorius muscle compared to the reference spectrum of a fully oxygenated
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sample (31) and also contains the simulated spectrum calculated using DSAP based on its determination of the cytochrome concentration. Via DSAP, the relative concentrations were calculated for cytochrome aa3 , cytochrome b, and cytochrome c as 43, 34, and 22%, respectively, compared to the published values of 56, 11, and 33%, respectively. Figure 5D shows values for the change in absorbance of rat liver mitochondria (32) and the simulated spectrum calculated with DSAP. The relative concentrations for cytochrome aa3 , cytochrome b, and cytochrome c were calculated via DSAP to be 52, 26, and 22%, respectively, compared to the published values of 43, 20, and 37%, respectively. DSAP was also applied to difference spectra obtained in this laboratory from rat liver mitochondria (Fig. 6). DSAP was used to calculate the relative concentrations of cytochrome aa3 , cytochrome b, and cytochrome c of 30, 57, and 13%, respectively. The results shown in Figs. 5 and 6 demonstrate the capacity of DSAP to confirm the experimental data. Calculations with DSAP also support the published qualitative descriptions of difference spectra. This application of multicomponent analysis can be applied to a variety of samples ranging from isolated organelles such as mitochondria to the complex mixtures of whole tissues or homogenates. DISCUSSION
Although multicomponent analysis of spectra is theoretically a relatively simple process, it has proven to be rather complex in practice. The major problems associated with analyzing biological systems by using spectral data include (1) the deconvolution of spectra
FIG. 6. Analysis of a difference spectrum of mitochondrial heme proteins with DSAP. Description of analysis is under Materials and Methods. (l) Values determined from spectrum of the mitochondrial sample; (—) model spectrum calculated with DSAP.
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containing oxidized and reduced heme proteins simultaneously present in a sample, and (2) turbidity or light scattering from nonheme components. Because the spectral data contain information about the oxidative status of a sample, precise quantitation of the heme proteins present is desirable. The PyrHo method presented here provides the necessary quantitation. This procedure is reproducible, and it also lessens the turbidity problem inherent in biological samples. Because a precise amount of heme proteins can be assigned to the spectra, the remaining amount of absorbance contributing to the spectra can be considered to be turbidity or light scattering. The fit of a calculated spectrum to the experimental data can be accomplished by two general procedures. Based on the chemical knowledge of the system, the experimenter can approach a solution through successive approximations of the quantities of heme protein species thought to be present in the mixture. Alternatively, the solver function of the spreadsheet program can be used to find an optimal solution based on the smallest sum of the least-squares differences between the calculated and experimental spectra. Initiation of the fitting process involves the use of estimations for the amounts of each heme protein present in the sample. Precise heme quantitation determined by the PyrHo method increases the accuracy of HSAP. If these judgments are not correct, the fitting process may continue to a least-square minimum that does not make chemical sense. It is often useful to constrain some of the parameters so that the results will give reasonable solutions (e.g., the exclusion of negative absorbancies). For a more detailed discussion of these and other mathematical problems of curve fitting, the reader is referred to Hendler and Shrager, (33) and Haaland and Thomas (34). Another challange in the curve fitting process is the amount of emphasis each data point contributes to the overall solution. This emphasis is known as the weighting factor. The most important indicators for heme protein oxidation are the differences seen in the a region (500 to 640 nm) of the spectra. The soret region (390 to 450 nm) provides additional information about the oxidation state of heme proteins. Since the soret absorbance is very high relative to the a region, weighting factors must be introduced to deemphasize this region’s contribution to the overall fitting process in the HSAP program. Therefore, two factors were introduced into the HSAP to assign appropriate weighting: (1) A factor correcting absorbancies obtained in the soret region which were scanned at a different dilution than those values obtained for the a and b peaks and (2) a factor to adjust the turbidity difference observed between the two regions. Reasonably good fits were produced when the HSAP was applied to spectra obtained from the literature.
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This success allowed us to roughly quantitate, from the spectral data given, the amount of heme proteins present in the original mixture. As an internal check of the accuracy of HSAP, spectra previously published by this laboratory were analyzed. These comparisons were similar to those obtained with HSAP and data published in the scientific literature. Although multicomponent analysis has been performed successfully in a variety of other applications, e.g., circular dichroism, electron paramagnetic resonance/nuclear magnetic resonance, or blood monitoring equipment, the number of components analyzed have been restricted because of the sheer complexity of the spectra obtained. Specialized equipment and software are sometimes necessary to deconvolute complex spectra so that individual components may be ascertained. The HSAP, on the other hand, can be used with a personal computer-based spreadsheet program and can deconvolute mixtures involving some of the different components of a complex heme protein spectrum. The basic concepts underlying HSAP can be applied, with minor modifications, to various situations. The HSAP, unlike much of the commercial spectrophotometric software available today, is easily modifiable, adapting to the constraints and conditions the experimenter employs (35). For instance, reflectance spectra that monitor skin or meat pigments can be deconvoluted to provide the amounts and types of heme proteins present in the sample by using HSAP. HSAP could provide a simple and noninvasive technique to measure the amount of vasodilation in the epidermis. Another practical application of HSAP would be the measurement of other chromophoric compounds such as chlorophylls and erythrocruorins, the invertebrate hemoglobins. Since these compounds also undergo a colorimetric change during oxidation of the metal nestled in the porphyrin ring, the amount of individual reduced or oxidized chlorophylls, for example, indicates the oxidative status of a plant sample. Plants and marine invertebrates also contain hydrocarbon chain chromophores, the carotenoids, and astaxanthins, respectively, that produce different absorbance spectra depending upon their oxidative status. In addition, the nonporphyrin proteins, anthocyanins and hemocyanins, present in plants and marine invertebrates can be followed spectrophotometrically. Their spectra also change with respect to the oxidation state of the bound metal. In combination with the total amount of chromophore present in a sample, the HSAP could thus be applied to a large variety of organisms and conditions. The multicomponent analysis capability of HSAP can be used in other applications. Inclusion of standard absorbance values from the infrared region in a modified HSAP, instead of the visible absorbancies for heme proteins, could be used to resolve components observed in infrared spectra. Alternatively, values that repre-
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sent the absolute difference in absorbance between oxidized and reduced cytochrome spectra can be used as reference points for deconvoluting difference spectra; an application outlined in this investigation (DSAP). Multicomponent analysis, by HSAP and related spreadsheet programming such as DSAP, represents a suitable technique for the quantitation of heme proteins in biological samples. HSAP, in particular, provides a reasonable estimate of the amount of oxidation and the type of heme protein involved in the oxidative process. Thus, HSAP is a simple, reproducible method for quantitating oxidative damage in biological samples. ACKNOWLEDGMENT We thank Ardelle Tappel for scientific and editorial assistance.
REFERENCES 1. Boyle, R. C., Tappel, A. L., Tappel, A. A., Chen, H., and Andersen, H. J. (1994) J. Agric. Food Chem. 42, 100–104. 2. Chen, H., Tappel, A. L., and Boyle, R. C. (1993) Free Radicals Biol. Med. 14, 509–517. 3. Sekuzu, I., and Takemari, S. (1972) in Electron and Coupled Energy Transfer in Biological Systems (King, T. E., and Klingenberg, M., Eds.), pp. 210, 346, 330, Dekker, New York. 4. Horie, S. (1968) in Structure and Function of Cytochromes (Okunuki, K., Kamen, M. D., and Sekuzu, I., Eds.), pp. 97–113, University Park Press, Baltimore. 5. Sato, R., Nishibayashi, H., and Ito, A. (1969) in Microsomes and Drug Oxidations (Gillette, J. R., Conney, A. H., Cosmides, G., Estabrook, R. W., Fouts, J. R., and Mannering, G. J., Eds.), pp. 111–132, Academic Press, New York. 6. Riggs, A. (1981) in Methods in Enzymology (Antonini, E., RossiBernardi, L., and Chiancone, E., Eds.), Vol. 76, pp. 5–28, Academic Press, New York. 7. Doss, M. (1968) Klin. Wochenschr. 46, 731–732. 8. Tentori, L., and Salvati, A. M. (1981) in Methods in Enzymology (Antonini, E., Rossi-Bernardi, L., and Chiancone, E., Eds.), Vol. 76, pp. 707–713, Academic Press, New York. 9. Chen, H., and Tappel, A. L. (1995) Free Radicals Biol. Med. 18, 949–953. 10. Andersen, H. J., Chen, H., Pellett, L. J., and Tappel, A. L. (1993) Free Radicals Biol. Med. 15, 37–48.
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11. Chen, H., Pellett, L. J., Andersen, H. J., and Tappel, A. L. (1993) Free Radicals Biol. Med. 14, 473–482. 12. Chen, H., and Tappel, A. L. (1993) Free Radicals Res. Commun. 19, 183–190. 13. Chen, H., and Tappel, A. L. (1993) J. Agric. Food Chem. 41, 1362–1367. 14. Berry, E. A., and Trumpower, B. L. (1987) Anal. Biochem. 161, 1–15. 15. International Committee for Standardization in Haematology (1978) J. Clin. Pathol. 31, 139–143. 16. Sekuzu, I. (1972) in Electron and Coupled Energy Transfer in Biological Systems (King, T. E., and Klingenberg, M., Eds.), pp. 207–220, Dekker, New York. 17. Wainio, W. W. (1970) The Mammalian Mitochondrial Respiratory Chain, pp. 254, 247, 281, Academic Press, New York. 18. Wharton, D., and Gibson, Q. (1968) J. Biol. Chem. 243, 702– 706. 19. Kinoshita, T., and Horie, S. (1967) J. Biol. Chem. 61, 26–34. 20. Sato, N., and Hagihara, B. (1968) J. Biol. Chem. 64, 723–726. 21. Gibson, O. H., and Wharton, D. C. (1968) in Structure and Function of Cytochromes (Okunuki, K., Kamen, M. D., and Sekuzu, I., Eds.), pp. 5–19, University Park Press, Baltimore. 22. Kindt, J., Woods, A., Martin, B. M., Cotter, R. J., and Osawa, Y. (1992) J. Biol. Chem. 267, 8739–8743. 23. Shikama, K., and Matsuoka, A. (1989) J. Mol. Biol. 209, 489– 491. 24. Tsuneshige, A., Imai, K., Hori, H., Tyuma, I., and Gotoh, T. (1989) J. Biochem. 106, 406–417. 25. Wittenberg, B. A. (1979) in Biochemical and Clinical Aspects of Oxygen (Caughey, W. S., Ed.), pp. 35–51, Academic Press, New York. 26. Williams, J. N. J. (1968) Biochim. Biophys. Acta 162, 175–181. 27. Andersen, H. J., Pellett, L., and Tappel, A. L. (1994) Chem.-Biol. Interact. 93, 155–169. 28. Knudsen, C. A., Tappel, A. L., and North, J. A. (1995) Free Radicals Biol. Med., in press. 29. Chance, B., and Schoener, B. (1966) J. Biol. Chem. 241, 4567– 4573. 30. Tappel, A. L. (1960) J. Cell. Comp. Physiol. 55, 111–126. 31. Jobsis, F. F. (1963) J. Gen. Physiol. 46, 905–928. 32. Tzagoloff, A. (1982) Mitochondria: The Electron Transfer Chain, pp. 89, Plenum, New York. 33. Hendler, R. W., and Shrager, R. I. (1994) J. Biochem. Biophys. Methods 28, 1–33. 34. Haaland, D. M., and Thomas, E. V. (1988) Anal. Chem. 60, 1193– 1202. 35. Cappas, C., Hoffman, N., Jones, J., and Young, S. (1991) J. Chem. Educ. 68, 300–303.
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Article No. 0015
Multicomponent Analysis of Heme Protein Spectra in Biological Materials James A. North, Dietrich Rein, and Al L. Tappel Department of Food Science and Technology, University of California, Davis, California 95616
Received August 1, 1995
Absorption spectra of heme proteins in a tissue homogenate contain information about the oxidation status of the biological sample. Deconvolution of absorption spectra can be used to quantitate the amount of individual heme proteins present in the mixture. The heme spectral analysis program (HSAP), a computer spreadsheet program, was used to quantitatively calculate values for heme proteins measured spectrally (390 to 450 and 500 to 640 nm) in tissue homogenates undergoing oxidation. The amount of oxidized heme proteins obtained by HSAP can be compared to other measurements of tissue oxidation. Precise quantitation of the amount of heme proteins present in a homogenate sample provided accurate assessment of the oxidized heme proteins calculated by HSAP. This quantitation was achieved through modification of existing pyridine hemochrome methods. Input into HSAP of the total heme protein content via the pyridine hemochrome value generated reproducible values for oxidized heme proteins. The program has broad potential as a multicomponent analysis tool. Modification of HSAP led to the development of a difference spectra analysis program (DSAP) which was used to quantitate the type and amount of heme proteins observed in mitochondrial difference spectra. In the present application, HSAP and DSAP provide methods for interpreting complex spectral information of multicomponent biological samples that undergo oxidation. q 1996 Academic Press, Inc.
In biological systems some oxidative events are beneficial, such as the detoxification of xenobiotics, whereas others are disastrous, e.g., the uncontrolled peroxidation of membrane lipids. Investigation into mechanistic details of oxidative processes in biological samples often requires quantitation of the damage observed in proteins, lipids, and other cellular components. Damage caused by oxidative reactions can be measured by a variety of assays that analyze the generation of prod-
ucts formed during the oxidative event. Of the many products generated, the oxidation of heme proteins represents a unique class of compounds that can be used as markers in the oxidative process. Heme proteins contain an iron–porphyrin complex capable of accepting unpaired electrons from various sources. Oxidation of the iron affects the energy state of the porphyrin ring and ultimately results in distinctly different spectra of the reduced and oxidized states. Spectral scans that read absorbance values across a range of visible wavelengths (390 to 640 nm) contain information about the oxidation state of individual heme proteins such as hemoglobin, myoglobin, or mitochondrial and microsomal cytochromes. Information derived from complex heme spectral scans obtained for samples originating from tissues, homogenates, or subcellular fractions can be obtained through comparison to reference spectra by multicomponent analysis. Although methods using multicomponent analysis are employed in laboratories to analyze the concentration of components from spectral data, the standard practice is to select specific wavelengths and, knowing the extinction coefficients of the different compounds at those wavelengths, solve the simultaneous equations generated. Existing methods of quantitation mostly measure irreversible oxidative products. A heme protein spectral analysis program previously developed in this laboratory is a method of deconvoluting composite spectra (500 to 640 nm) and obtaining molar ratio values of heme proteins in tissue samples (1). This heme protein spectral analysis program, measuring reversible and irreversible damage to heme proteins, uses a spreadsheet program that analyzes absorbancies at many points in the spectrum rather than a few wavelengths and a combination of regression analysis and successive approximations. Tissue homogenate samples contain a high amount of turbidity. This can be reduced by subtracting an appropriate level of light scatter from a background scan prior to acquisition of spectral data. 115
0003-2697/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Several layers of parafilm have been found to be adequate under most circumstances to simulate some of the background turbidity. Light scatter is not linear with respect to wavelength; therefore, a turbidity calculation had been introduced to the heme protein spectral analysis program (1, 2). In the present study, absorbance values corresponding to the gamma peaks of the various heme proteins (390 to 450 nm) have been added to the previous program and form a major contribution to the new heme spectra analysis program (HSAP).1 With the previous program (1) we could adequately analyze reduced hemoglobin species. However, measurement of oxidized heme proteins presented a unique problem in unambiguously assigning individual spectra. Oxidized heme proteins of the mitochondria, microsomes, the oxidized, partially denatured hemoglobin compound hemichrome, and methemoglobin produce spectra that closely resemble each other in the region from 500 to 640 nm. One solution was to include spectral data in HSAP for these oxidized heme proteins arising from the strongly absorbing Soret region containing the g peaks. In this region, peak maxima for oxidized heme components are sufficiently different for discrimination with HSAP (methemoglobin, 406 nm; hemichrome, 416 nm; combined oxidized mitochondria including cytochrome aa3, cytochrome b, and cytochrome c, 414 nm; and combined oxidized microsomes including cytochrome P450 and cytochrome b5, 425 nm). Thus, the recording of g peaks allows additional assignment of oxidized heme proteins originating from various sources, including mitochondrial or microsomal cytochromes that are normally hidden in spectra because of the overwhelming amount of hemoglobin present in nonperfused tissue homogenates. HSAP was also constrained to solve spectra obtained through oxidation of heme proteins by recognizing chemical reactions that can occur in the samples. For example, fully oxygenated homogenate samples should not contain reduced hemoglobin but instead oxyhemoglobin, so the optimization portion of the HSAP did not include deoxygenated, reduced hemoglobin in its calculations. Further refinements of HSAP based on the chemical reactions possible in the homogenate can be used when solving multicomponent spectra. These refinements may include fixing the amount of mitochondria and microsomes present in a given sample. Values for the amounts of organelles in a particular tissue homogenate can be estimated by reviewing the literature (3–5). A further aid in analyzing the spectra to be deconvoluted and quantitated by the modified HSAP is the 1 Abbreviations used: HSAP, heme spectra analysis program; PyrHo, pyridine hemochrome; DSAP, difference spectra analysis program.
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use of the pyridine hemochrome (PyrHo) technique to quantify the total amount of heme proteins in highly turbid homogenate samples (6, 7). Routine hemoglobin measurements often utilize cyanide solutions to convert hemoglobin to the cyanomethemoglobin derivative (8). Turbidity present in tissue homogenates interferes with measurements of cyanomethemoglobin. PyrHo, however, is measured by difference spectra so that turbidity does not contribute to the final calculations. Therefore, PyrHo values were used in HSAP as an independent fixed parameter. This PyrHo method has two advantages, the minimization of interference due to turbidity in tissue homogenates and the vivid illustration of the large amount of hemoglobin present in samples that can obscure spectral absorbance from cytochromes and other heme proteins. To aid in the deconvolution of the adjoining a and b peaks of cytochromes of organelles, the difference spectra analysis program (DSAP) was developed in the present investigation. Difference spectra are routinely used to identify the heme components of organelles such as mitochondria. Spectral values of oxidized cytochrome standards were subtracted from the spectral values of reduced cytochrome standards to provide a reference for this multicomponent difference analysis. The dominant absorbance peaks, or a peaks, observed in difference spectra represent absorbance changes for cytochrome aa3 (605 nm), cytochrome c (550 nm), and cytochrome b (560 nm, usually as a shoulder of the cytochrome c peak). The b peaks for these components appear near 518 nm; the soret bands appear in the 430 to 450 nm region. Although enough information is available to provide evidence for the type of heme protein present in a mitochondrial difference spectra, quantitation of the individual components by using the entire difference spectra routinely has not been done. This multicomponent analytical technique facilitates the quantitation of individual heme proteins present in a mitochondrial sample. This paper demonstrates the use of HSAP and its sister program DSAP to measure changes in the oxidative state of heme proteins in tissues and other biological systems. The programs can be relevant to some aspects of clinical problems associated with tissue injury that involve free radical reactions. MATERIALS AND METHODS
Chemicals. Rat and human hemoglobins and sodium dithionite were purchased from Sigma Chemical Co. (St. Louis, MO); pyridine and other solvents were from Fisher Scientific (Pittsburgh, PA). Animals. Tissue homogenates were prepared from male Sprague–Dawley rats fed synthetic diets as described previously (2, 9). Tissue homogenates in Krebs–Ringer phosphate buffer (10% w/v) were trans-
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ferred to glass serum bottles and closed with rubber stoppers. The homogenates were incubated in a Gyrotory water bath shaker (New Brunswick Scientific Co., Inc., New Brunswick, NJ) at 377C with continuous shaking (180 cycles/min). Spectrophotometric measurement of heme proteins from tissue homogenates. The absorbance spectra of tissue homogenates were obtained with a Beckman DU-50 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). Tissue homogenates were transferred to a cuvette with a light path of 10 mm containing equal volume buffer (final tissue concentration: 50 mg/ ml) and mixed. Parafilm, representing turbidity, was used as a background to subtract some of the absorbance caused by turbidity inherent in tissue homogenates. Samples were scanned from 390 to 450 nm (gregion) at a 101 dilution and from 500 to 640 nm (a and b regions). Absorbance vs wavelength at 2-nm intervals was automatically recorded by a scan program in the spectrophotometer. Spectral data obtained for fresh homogenates and for incubant samples were transferred to the spreadsheet computer program. Analysis of absorbance spectra of heme proteins of tissue homogenates. HSAP is a spreadsheet program written with Excel 5.0 (Microsoft Corp., Redmond, WA) that contains the micromolar absorbance values from 390 to 450 nm and from 500 to 640 nm of the visible spectra of individual standard heme proteins. The analysis is based on the knowledge that the absorbance spectrum of a mixture of heme proteins closely obeys Beer’s law and is the sum of the spectra of the individual heme proteins including any contribution from turbidity. Quantitation is achieved by matching a spectrum calculated from a combination of standard spectra with the experimental spectrum through successive approximations. These calculations can be automated through the use of the spreadsheet program solver or optimization function (Frontline Systems, Inc., Incline Village, NV). Development and applications of multicomponent analysis programs have been previously described (10–13). Specific details about a heme protein spectra analysis program can be obtained from Chen and Tappel (13). Modifications described in the present study are: (1) 2-nm intervals from 390 to 450 and from 500 to 640 were entered into HSAP; (2) turbidity contribution to the spectrum absorbance was calculated by using the light scattering formula: Turbidity Å alb, where a and b represent constants specific to that particular homogenate mixture at the wavelength l; and (3) total heme proteins, measured by the pyridine method, were added as a constraint for HSAP. Total heme quantification by the pyridine hemochrome method. Conversion of heme proteins to their PyrHo derivative was used to determine the total amount of heme proteins present in the tissue homoge-
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FIG. 1. Pyridine hemochrome standard curve generated using cyanomethemoglobin as a standard reference. The concentration per gram rat hemoglobin determined by the cyanomethemoglobin method was plotted against the absorbance difference per gram rat hemoglobin obtained by the PyrHo technique and is shown as data points (j). The regression line drawn through the points has a slope Å 53.8 mM heme per unit of absorbance difference, R 2 Å 0.98.
nates (14). Briefly, an equal volume of a stock solution containing 200 mM NaOH, 40% vol pyridine and 0.8 mM K3Fe(CN)6 was added to an aliquot of the tissue homogenate and vortexed. This oxidized sample was used to blank the spectrophotometer for the subsequent scan of the reduced sample. Sodium dithionite at a final concentration of 0.02 mM was added to this mixture to reduce hemichromes to hemochromes and after 3 min the maximum and minimum absorbancies were obtained from the interval between 535 to 560 nm. The minimum absorbance around 539 nm (a – b valley) was subtracted from the maximum absorbance around 555 nm (a peak). An extinction coefficient of 19.2 mM01 for PyrHo (see calibration below) was used to calculate the molar tissue heme concentration for each sample. The molar extinction coefficient for PyrHo was obtained by combining and calibrating two independent methods used to quantify total heme components. Literature values for the molar extinction coefficient of the PyrHo spectrum vary greatly with a range between 19 and 24 mM01. We calibrated the PyrHo assay for tissue homogenates against the generally accepted cyanomethemoglobin technique for quantitation of heme compounds (3, 15) using a rat hemoglobin standard (Fig. 1; R 2 Å 0.98). Subjecting the hemoglobin standard solution to PyrHo analysis as outlined above resulted in an extinction coefficient of 19.2 mM01. Both the cyanomethemoglobin and PyrHo methods produced similar results when a low turbidity standard was used. The PyrHo method, however, is far less susceptible to interference by trubidity since it is based on difference spectra analysis. It was therefore used to quantify total heme compounds in the homogenate samples.
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Analysis of difference spectra of heme proteins of mitochondria. DSAP is a spreadsheet program written with Excel 4.0 (Microsoft Corp.) and contains millimolar absorptivities from 500 to 640 nm of the visible spectra of the major heme containing proteins present in mammalian mitochondria, cytochromes aa3 , cytochrome b, and cytochrome c. Adding other heme protein spectra, e.g., cytochrome c1 , would not alter the analysis greatly because the additional spectra would fit well within one of the three major species included in the DSAP. Although the maximum and minimum absorbancies may be different for different cytochromes within the same family, we concluded that the various difference spectra obtained by subtracting the absorptivity values of the oxidized form of the cytochrome from the reduced form were similar. The theory for analysis and quantitation is similar to that for HSAP except that values are obtained by successive approximations of difference spectra in which the oxidized spectral absorbancies have been subtracted from the reduced spectral absorbancies of heme proteins. Cytochrome millimolar absorptivities were calculated from spectra obtained from the literature (3–5, 16–21). Spectra from the literature were scanned and digitized using software available from the NIH (Image 1.49, NIH, 1989). Statistical evaluation. Data are expressed as means { SE. All observations were performed with at least triplicate samples. RESULTS
Application of HSAP to Heme Protein Spectra Figure 2A shows spectral changes of heme-containing proteins during oxidative reactions. Before incubation, the dominant absorbance peaks appearing around 530 and 575 nm indicate that oxyhemoglobin is the major component. The location of the soret band at around 415 nm supports this finding. After 2 h incubation, spectral changes occurred in both regions showing the formation of methemoglobin and other oxidized heme proteins. Standard spectra of oxidized heme proteins are shown in Fig. 2B. HSAP Analysis of Published Spectra To ascertain the reproducibility and accuracy of HSAP with respect to the identification and tentative quantitation of hemoglobin species, published spectra were subjected to multicomponent analysis. The results are shown in Fig. 3. Figure 3A shows absorbance values from a spectrum of human hemoglobin (22) reduced by addition of sodium dithionite and the model spectrum calculated with HSAP based on its determination of heme protein concentrations. HSAP calculated the concentration of hemoglobin to
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FIG. 2. Spectral changes of heme proteins. (A) The changes in liver tissue homogenates incubated at 377C. Soret peaks are 1/10 original values (380 to 450 nm). Top spectra, 0 h; bottom spectra, 2 h. (s) Spectral absorbance data points; (—) spectra calculated with HSAP where, in the 500 to 640 nm region, it is obscured by the absorbance data points. (—) Lines under spectra show turbidity calculated with HSAP. (B) The individual and composite standard spectra that can contribute to the oxidized spectra observed after 2 h incubation. The lines represent (—) methemoglobin, (---) hemichrome, and (– –) oxidized heme proteins of mitochondria.
be approximately 2.7 mM compared to the published value of 3.0 mM. HSAP analyses were performed independently of the values found in the literature, thus the agreements on concentrations show the usefulness of this program. Kindt et al. (22) also measured methemoglobin, the oxidized form of hemoglobin, and the absorbance values are shown in Fig. 3B along with the model spectrum calculated with HSAP. On the basis of its determination of heme protein concentrations, HSAP calculated the concentration of methemoglobin to be approximately 3.03 mM compared to a published value of 3.0 mM. Figure 3C shows absorbance values from a spectrum of oxygenated sperm whale myoglobin (23) and the model spectrum calculated with HSAP based on the determination of heme protein concentrations. Myoglobin and metmyoglobin spectra resemble hemoglobin and methemoglobin spectra such that HSAP calculated the concentration to be approximately 9.9 mM compared to the published value of 10 mM. Shikama and
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chromes of the mitochondria as the heme proteins responsible for the spectral data in the proportions expected in rat heart (26), approximately 38 and 62%, respectively. The results shown in Fig. 3 demonstrate the ability of HSAP to supply good calculated fits to the experimental data. HSAP approximations also support the published qualitative description of the spectra and is in accord with the expected biochemistry. The published spectra used contain only one or two hemoglobin species whereas tissue and homogenate samples represent complex spectra containing multiple heme protein mixtures. Heme Quantification in Tissues
FIG. 3. Analysis of absorbance spectra of heme proteins from the literature with HSAP. Description of analysis is under Materials and Methods. Sources are cited under Results. (l) Values determined from spectra from the literature; (—) model spectra calculated with HSAP. The (---) line under the spectra is the absorbance calculated for turbidity with HSAP.
Matsuoka (23) also measured metmyoglobin, the oxidized form of myoglobin, and both the absorbance values and the calculated spectra are shown in Fig. 3D. Via HSAP, the metmyoglobin concentration was calculated to be approximately 9.3 mM compared to the published value of 10 mM. Figure 3E shows molar absorptivity values from a spectrum of oxygenated lugworm hemoglobin (24) and the spectrum calculated with HSAP. Since the millimolar absorptivities were used as the experimental absorbance values to calculate the millimolar concentrations of heme proteins in HSAP, the expected concentration of oxygenated hemoglobin should be approximately 1000 mM. HSAP calculated a concentration of HbO2 in the lugworm to be 961 mM, in good agreement with the expected value. Figure 3F shows absorbance values from a spectrum of isolated adult rat heart cells under anaerobic conditions (25), and the simulated spectrum calculated with HSAP. Because the published spectrum provided only relative measurement of absorbance, HSAP could only be used to provide qualitative information. In spite of these limitations, HSAP matched the experimental spectrum using only deoxymyoglobin and reduced cyto-
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Measuring the amount of heme protein in blood samples routinely consists of converting all forms of the heme into a common spectrally defined derivative, such as cyanomethemoglobin (8, 15). The turbidity present in tissue homogenates, however, directly interferes with the measurements of cyanomethemoglobin. Similar to the cyanomethemoglobin method, the pyridine hemochrome technique does not depend on the initial ligand state of hemoglobin to produce a spectrally defined common derivative (6). Turbidity, however, does not interfere with PyrHo measurements. The extinction coefficient of 19.2 mM01cm01 was used to convert PyrHo values to heme protein molar concentrations for determining the amount of heme compounds in a biological system. Addition of an alkaline solution containing pyridine and the reducing agent sodium dithionite stoichiometrically converts heme iron porphyrins to hemochromes in the heme mixtures tested. The hemochrome structure has a sharp absorption maximum around 556 nm whereas the corresponding oxidized porphyrin–pyridine adduct, hemichrome, has a diffuse spectrum with maxima near 534 and 556 nm. The absorption difference between reduced and oxidized PyrHo minimizes spectral interference due to turbidity and thus can be used to quantify the amount of PyrHo present in the heme protein samples (7). The calibration of PyrHo using rat hemoglobin or tissue homogenates resulted in a linear relationship between the amount of heme calculated to be present in the solution and the difference in absorbance between reduced and oxidized pyridine adducts (Fig. 1, rat Hb; R 2 Å 0.98) (Fig. 4, rat liver homogenate; R 2 Å 0.99). The results obtained with liver homogenates prepared from basal diet-fed animals showed that the PyrHo method produced consistent heme protein values (Table 1). When the liver homogenate at a sample concentration of 0.05 g/ml was assayed in several different preparations, the values were consistent within groups as well as between groups. When the liver homogenate concentration was varied, the total heme pro-
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Pyridine Hemochrome Replication
FIG. 4. Heme content of liver samples as a function of homogenate concentration. Total heme concentration was determined by the pyridine hemochrome method outlined under Materials and Methods. (j) Data points represent results from three different rat livers. The regression line has a slope of 0.372 mM heme/g liver; R 2 Å 0.99.
tein content calculated by the PyrHo technique was similar; 0.29 { 0.01 mmol heme protein/kg liver (Table 2, average { SE). These results indicate that PyrHo can provide reproducible quantitation of the total amount of heme proteins in tissue homogenates. Another assay used to support the accuracy of the PyrHo method is a recovery experiment. Hamster red blood cells were assayed for total heme concentration in the presence or absence of a known amount of rat hemoglobin standard. Even when the concentration of the red blood cell was varied, all of the hemoglobin standard could be accounted for in the mixture (Table 3). Although the PyrHo method gives reproducible results, the total amount of heme proteins will vary from tissue to tissue. This variance depends mainly upon the amount of perfusion and if the organ were subjected to prior storage and experimentation. However, since the total amount of heme proteins is small in comparison to the total amount of proteins in the entire sample,
TABLE 1
Pyridine Hemochrome Replication
Tissue concentration (mg/ml)
Total heme value (mmol/kg tissue)
50 50 25 25 12.5 12.5
0.30 0.28 0.24 0.29 0.29 0.30
Average SE
0.29 0.01
hemoglobin can serve as a marker for oxidation independent of its actual concentration. In fact, when tissue homogenates containing a wide divergence in the amounts of hemoglobin were allowed to oxidize for 2 h, the percentages of oxidized heme proteins measured by HSAP were not significantly different (duplicate samples of rat heart homogenates containing 19, 21, and 91 mM PyrHo produced 82, 80, and 86% oxidized heme protein values, respectively). The absolute amount of oxidized heme proteins differed between the various preparations (16, 17, and 78 mM oxidized heme proteins, respectively). Thus, comparisons between different preparations of the same tissue, the same tissue from different animals, or between different tissues can effectively be normalized and subjectively interpreted (for examples, see Refs. 1, 10, 11, 27, 28). Application of DSAP to Mitochondrial Difference Spectra Figure 5 represents difference spectra obtained from the literature and analyses of the spectra calculated with DSAP. The calculated spectra confirm the experimental data in each case. Turbidity does not interfere in these spectra because difference spectra are used to cancel out turbidity. Another advantage of the DSAP is the emphasis of the cytochrome b peak contribution
mmol heme/kg tissue TABLE 3 Group:Sample No.
Total heme value
Group average { SE
A:1 A:2 A:3 A:4 B:1 B:2 B:3 C:1 C:2 C:3
0.24 0.24 0.23 0.32 0.28 0.29 0.30 0.26 0.28 0.34
0.27 { 0.02
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Hemoglobin sample 0.29 { 0.01
0.29 { 0.02
Red blood cell Hemoglobin standard Red blood cell plus hemoglobin standard Recovery
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0.1% red blood cell by volume
0.2% red blood cell by volume
24 { 1.2 64
49 { 2.4 64
91 { 0.4
114 { 1.1
104%
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MULTICOMPONENT ANALYSIS OF HEME SPECTRA
FIG. 5. Analysis of difference spectra of mitochondrial heme proteins from the literature with DSAP. Description of analysis is under Materials and Methods. Sources are cited under Results. (l) Values determined from spectra from the literature; (—) model spectra calculated with DSAP.
to the overall spectrum. Even in the presence of large amounts of cytochrome c, DSAP reproducibly assigned values for cytochrome b in samples that contained both cytochrome c and cytochrome b. Figure 5A shows a difference spectrum of pigeon heart mitochondria (29) and the model difference spectrum calculated with DSAP based on its determination of cytochrome concentrations. Using DSAP, relative concentrations were calculated for cytochrome aa3 , cytochrome b, and cytochrome c at 52, 4, and 44%, respectively, of the total cytochrome content. Despite the absence of compositional data, the calculated spectrum was in accord with the experimental spectrum. Figure 5B shows a difference spectrum of crab flexor muscle (30) and the simulated spectrum calculated with DSAP. The percentage compositions of cytochrome aa3 , cytochrome b, and cytochrome c were calculated via DSAP to be 62, 23, and 15%, respectively, compared to the published values of 72, 7, and 21%, respectively. Figure 5C shows optical density changes in the difference spectrum of anoxic toad sartorius muscle compared to the reference spectrum of a fully oxygenated
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sample (31) and also contains the simulated spectrum calculated using DSAP based on its determination of the cytochrome concentration. Via DSAP, the relative concentrations were calculated for cytochrome aa3 , cytochrome b, and cytochrome c as 43, 34, and 22%, respectively, compared to the published values of 56, 11, and 33%, respectively. Figure 5D shows values for the change in absorbance of rat liver mitochondria (32) and the simulated spectrum calculated with DSAP. The relative concentrations for cytochrome aa3 , cytochrome b, and cytochrome c were calculated via DSAP to be 52, 26, and 22%, respectively, compared to the published values of 43, 20, and 37%, respectively. DSAP was also applied to difference spectra obtained in this laboratory from rat liver mitochondria (Fig. 6). DSAP was used to calculate the relative concentrations of cytochrome aa3 , cytochrome b, and cytochrome c of 30, 57, and 13%, respectively. The results shown in Figs. 5 and 6 demonstrate the capacity of DSAP to confirm the experimental data. Calculations with DSAP also support the published qualitative descriptions of difference spectra. This application of multicomponent analysis can be applied to a variety of samples ranging from isolated organelles such as mitochondria to the complex mixtures of whole tissues or homogenates. DISCUSSION
Although multicomponent analysis of spectra is theoretically a relatively simple process, it has proven to be rather complex in practice. The major problems associated with analyzing biological systems by using spectral data include (1) the deconvolution of spectra
FIG. 6. Analysis of a difference spectrum of mitochondrial heme proteins with DSAP. Description of analysis is under Materials and Methods. (l) Values determined from spectrum of the mitochondrial sample; (—) model spectrum calculated with DSAP.
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containing oxidized and reduced heme proteins simultaneously present in a sample, and (2) turbidity or light scattering from nonheme components. Because the spectral data contain information about the oxidative status of a sample, precise quantitation of the heme proteins present is desirable. The PyrHo method presented here provides the necessary quantitation. This procedure is reproducible, and it also lessens the turbidity problem inherent in biological samples. Because a precise amount of heme proteins can be assigned to the spectra, the remaining amount of absorbance contributing to the spectra can be considered to be turbidity or light scattering. The fit of a calculated spectrum to the experimental data can be accomplished by two general procedures. Based on the chemical knowledge of the system, the experimenter can approach a solution through successive approximations of the quantities of heme protein species thought to be present in the mixture. Alternatively, the solver function of the spreadsheet program can be used to find an optimal solution based on the smallest sum of the least-squares differences between the calculated and experimental spectra. Initiation of the fitting process involves the use of estimations for the amounts of each heme protein present in the sample. Precise heme quantitation determined by the PyrHo method increases the accuracy of HSAP. If these judgments are not correct, the fitting process may continue to a least-square minimum that does not make chemical sense. It is often useful to constrain some of the parameters so that the results will give reasonable solutions (e.g., the exclusion of negative absorbancies). For a more detailed discussion of these and other mathematical problems of curve fitting, the reader is referred to Hendler and Shrager, (33) and Haaland and Thomas (34). Another challange in the curve fitting process is the amount of emphasis each data point contributes to the overall solution. This emphasis is known as the weighting factor. The most important indicators for heme protein oxidation are the differences seen in the a region (500 to 640 nm) of the spectra. The soret region (390 to 450 nm) provides additional information about the oxidation state of heme proteins. Since the soret absorbance is very high relative to the a region, weighting factors must be introduced to deemphasize this region’s contribution to the overall fitting process in the HSAP program. Therefore, two factors were introduced into the HSAP to assign appropriate weighting: (1) A factor correcting absorbancies obtained in the soret region which were scanned at a different dilution than those values obtained for the a and b peaks and (2) a factor to adjust the turbidity difference observed between the two regions. Reasonably good fits were produced when the HSAP was applied to spectra obtained from the literature.
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This success allowed us to roughly quantitate, from the spectral data given, the amount of heme proteins present in the original mixture. As an internal check of the accuracy of HSAP, spectra previously published by this laboratory were analyzed. These comparisons were similar to those obtained with HSAP and data published in the scientific literature. Although multicomponent analysis has been performed successfully in a variety of other applications, e.g., circular dichroism, electron paramagnetic resonance/nuclear magnetic resonance, or blood monitoring equipment, the number of components analyzed have been restricted because of the sheer complexity of the spectra obtained. Specialized equipment and software are sometimes necessary to deconvolute complex spectra so that individual components may be ascertained. The HSAP, on the other hand, can be used with a personal computer-based spreadsheet program and can deconvolute mixtures involving some of the different components of a complex heme protein spectrum. The basic concepts underlying HSAP can be applied, with minor modifications, to various situations. The HSAP, unlike much of the commercial spectrophotometric software available today, is easily modifiable, adapting to the constraints and conditions the experimenter employs (35). For instance, reflectance spectra that monitor skin or meat pigments can be deconvoluted to provide the amounts and types of heme proteins present in the sample by using HSAP. HSAP could provide a simple and noninvasive technique to measure the amount of vasodilation in the epidermis. Another practical application of HSAP would be the measurement of other chromophoric compounds such as chlorophylls and erythrocruorins, the invertebrate hemoglobins. Since these compounds also undergo a colorimetric change during oxidation of the metal nestled in the porphyrin ring, the amount of individual reduced or oxidized chlorophylls, for example, indicates the oxidative status of a plant sample. Plants and marine invertebrates also contain hydrocarbon chain chromophores, the carotenoids, and astaxanthins, respectively, that produce different absorbance spectra depending upon their oxidative status. In addition, the nonporphyrin proteins, anthocyanins and hemocyanins, present in plants and marine invertebrates can be followed spectrophotometrically. Their spectra also change with respect to the oxidation state of the bound metal. In combination with the total amount of chromophore present in a sample, the HSAP could thus be applied to a large variety of organisms and conditions. The multicomponent analysis capability of HSAP can be used in other applications. Inclusion of standard absorbance values from the infrared region in a modified HSAP, instead of the visible absorbancies for heme proteins, could be used to resolve components observed in infrared spectra. Alternatively, values that repre-
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sent the absolute difference in absorbance between oxidized and reduced cytochrome spectra can be used as reference points for deconvoluting difference spectra; an application outlined in this investigation (DSAP). Multicomponent analysis, by HSAP and related spreadsheet programming such as DSAP, represents a suitable technique for the quantitation of heme proteins in biological samples. HSAP, in particular, provides a reasonable estimate of the amount of oxidation and the type of heme protein involved in the oxidative process. Thus, HSAP is a simple, reproducible method for quantitating oxidative damage in biological samples. ACKNOWLEDGMENT We thank Ardelle Tappel for scientific and editorial assistance.
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