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Determination of iron in heme compounds
ANALYTICAL
BIOCHEMISTRY
11,
Determination
159-163
of
(1965)
Iron
in
Heme
Compounds
I. Hemin ALAN From
the Department University
D.
ADLER
AND
of Biology and of Pennsylvania, Received
PHILIP
John Harrison Philadelphia, July
GEORGE Laboratory Pennsylltania
of Chemistry,
7, 1964
Calorimetric determination of the iron in natural products by means of o-phenanthroline has been described by several authors. This literature is reviewed in detail in the comprehensive monograph on phenanthroline analytical methods by Smith and Richter (1) along with most of the pertinent physical and chemical data. The determination of iron specifically in hemin by such methods was first given by Drabkin (2). Drabkin’s method consists in decomposition of the hemin by H,O, in alkaline media, heating to remove the peroxide, acidification and digestion with HCI, buffering to approximately pH 4 with saturated ammonium acetate, reducing from ferric to ferrous iron with ascorbic acid, forming the o-phenanthrolinc complex with excess reagent, and finally calorimetrically determining the optical density at about 500 my (2 cm,kf = 11.05) against a proper blank. This procedure requires several hours for the determination of a single sample. As it is frequently necessary to obtain values for the iron content of iron porphyrins in physical studies of these materials, we have modified the above procedure to provide a somewhat more rapid and convenient determination. METHODS
Deionized water distilled from alkaline permanganate is used throughout this procedure for making up solutions and the final rinsing of all glassware. After initial cleaning the glassware is routinely cleaned with hot tap water and detergent and then rinsed with the distilled deionized water and allowed to dry. The pyridine used should be fresh reagent or spectral grade (or its equivalent). Pyridine exposed to air is observed to yellow very slowly due to the formation of pyridine oxide, etc. The aqueous 2.0 M solution is stable, however, for several months. A hemin sample (10.00 to 15.00 mg) is weighed out on a cleaned and tared glass microscope slide cover slip on a microbalance. The slip is picked up with tweezers and the hemin is washed via a small funnel into 159 01965
by Academic
Press Inc.
160
ADLER
AND
GEORGE
a 25.0-ml volumetric flask with 2.0 M aqueous pyridine solution. The solution is then adjusted to volume with the pyridine solution to form a hemin stock solution. A LOO-ml aliquot of the hemin stock solution is transferred to a 25.0-ml volumetric flask and allowed to react with 0.2 ml of 30% H202 (Merck Superoxal) until gas evolution ceases. The flask is gently agitated and swirled for the 5 min required for the complete decomposition. Then several aliquots of solid recrystallized ascorbic acid sufficient to cover the tip of a microspatula (approximately 0.5 mg/aliquot) are added to the decomposition mixture until no gas evolution occurs upon addition of a further aliquot (generally 3 to 4 aliquots are required). Then 2.0 ml of 0.1% aqueous o-phenanthroline (G. Frederick Smith Chemical Co.) is added and the solution brought up to the mark with distilled deionized water. After allowing 15 min for color development, the optical density is spectrophotometrically determined with a Beckman DU against a blank at 509 mP and the iron content calculated from an c1 Cmmdl= ll.O-the average of the values given by several authors for the tris-Fe%-phenanthroline complex is P C*nmJf= 11.1 at 510 rnp (1). The optical density is rechecked after another 5 min. Occasionally a sample fails to recheck to within 0.001 optical density. Such samples should be discarded and another determination made from the hemin stock solution. The blank comprises all the reagents but the hemin and should be carried through the procedure simultaneously with the hemin sample. However, distilled water blanks may also be used, since the LLdetermination” blank vs. water usually gives readings less than 0.001 optical density. Mohr’s salt, Fe (NH,) 2 (SO,) 2 -6 H,O, was used as the primary standard to check, calibrate, and determine the 8 Cm,M for this procedure. Freshly recrystallized reagent-grade salt was employed in order to avoid the problem of efflorescence (3). Beer’s law was tested for the tris-Fen-ophenanthroline complex under the conditions of this method with Mohr’s salt over the range of optical densities 0.010 to 0.850 and found to be obeyed. This experiment gave the extinction coefficient quoted above. The following changes in procedure were found not to alter significantly the blank, the requisite conditions, the accuracy, or the precision of the method: variation of temperature from 15 to 35”C, variation of pyridine stock solution concentration from 1.0 to 4.0 M, variation of the H,O, employed from 0.1 to 0.4 ml, variation of decomposition time from 2.5 to 10 min, variation of the solid ascorbic acid used from half to treble the usual amount, variation of the o-phenanthroline solution added from 1.5 to 3 ml, variation of color development time from 10 min to 0.5 hr, and variation of wavelength of measurement from 508 to 510 mp. Further,
IRON
IN
HEME
161
COMPOUNDS
the 15-min value of the optical density of both a set of hemin and Mohr’s salt samples were found to be stabIe for five days, after which time they were discarded. Several other oxidants and reductants were also tested for this procedure-H,O, and ascorbic acid remained the most convenient reagents, although Cl, or ClO, and hydroxylamine salts or hydrazine were also found to be suitable. As 25 determinations can be rapidly made upon a single weighing, the precision of the method can be very high. Agreement between such determinations and also between triplicate weighed samples are found to be limited to the precision and accuracy of the Beckman DU spectrophotometer (which is maximally about 0.570). The sample weights employed give optical densities in the range 0.200 to 0.500 where the spectrophotometer gives the best’ compromise between sensitivity and accuracy (4). With practice the entire determination can be carried out in about onehalf hour. The sensitivity of the procedure can be extended by weighing out 0.40 to 0.60 mg of hemin and transferring directly to the decomposition vessel with 1.0 ml of 2.OM aqueous pyridine. It may be further extended by a factor of about 20 by the use of bathophenanthroline and IO-cm light path length cells. The method has been found equally applicable to several other iron porphyrin complescs than protohemin and also to other metalloporphyrins such as the Cu, Co, etc. derivatives. RESULTS
Iron determinations were made upon recrystallized and crude horse blood hemin, prepared according to Fischer (5)) and, also, on four commercial samples purchased from Eastman Kodak, L. Light & Co., Sigma Chemical Co., and California Biochemical Corp. Duplicate weighings were made for each sample, five determinations were performed with each hemin stock solution, and the ten values were averaged. The average % Fe and 70 expected Fe for each sample are given in Table 1. The average deviations for average ‘$% Fe’s are all less than -~0.0170. However, as this represents the precision and accuracy of the DU spectrophotometer TABLE IRON Source
CONTENT
of hemin
OF SIX
sample
Recrystallized (horse blood) Crude (horse blood) Eastman Kodak L. Light & Co. Calif. Biochem. Corp. Sigma Chem. Co.
1
DIFFERENT
HEMIN 70 Fe
8.31 7.20 8.15 8.01 s.01 7.89
S.UVPI,ES % expected
97.1 s4.0 95.5 93.5 93.5 93.1
Fe
162
ADLER
AND
GEORGE
and not of the chemical procedure, its merit as an error estimate is somewhat limited. The theoretical iron content for hemin is 8.5770 by weight. The procedure gave 99.9% of the expected iron content for a single control Mohr’s salt sample run simultaneously with these samples. DISCUSSION
Microscope cover slips and the washing transfer are employed to avoid the problem of hemin samples sticking to surfaces such as weighing paper, etc. As the cover slips weigh only about 100 mg, taring preserves the sensitivity of the microbalance. The presence of pyridine in the decomposition reaction mixture prevents the formation of colloidal iron sols as in the Drabkin procedure. Further, combination of the pyridine and ascorbic acid in the final solution serves to buffer it in the optimum pH range for this determination (1) without addition of any further buffering agents. Though this method gives the same accuracy and precision as the Drabkin method, it is faster and more convenient. Furthermore, the only reagents employed are so low in iron content that in the amounts employed in the determination they contribute no appreciable color to the blank. The 2.0 M pyridine and the 0.1% o-phenanthroline solution are the only required stock solutions. These are readily prepared and both are stable for months (and even for longer under refrigeration and in appropriately sealed containers). No heating steps are required as in previous methods. Finally the method is such that the limiting sources of systemat.ic error are in the initial weighing and in the optical density reading. This is easily further limited to the optical density reading by the proper use of a good microbalance. The interpretation of the wide variation in the results obtained for the six different hemin samples can only be given without speculation when these results are compared with further analytical studies on the C, H, N, 0, and Cl contents of these same samples. As we have performed such further studies, these data will be reviewed and discussed in a separate paper. SUMMARY
An accurate, precise, rapid, and convenient method for the spectrophotometric determination with o-phenanthroline of the iron content of hemin is detailed. The limitations, modifications, and extensions of the method are discussed. The application of the method to six different solid hemin samples is presented to exemplify the utility of the method. This work was supported
ACKNOWLEDGMENT by Public Health Service Grant AM-03187.
IRON
IN
HEME
COMPOUNDS
163
REFERENCES 1. SMITH, G. F. AIVD RICHTER, F. I’., “Phenanthroline and Substituted Phenanthroline Indicators.” G. Frederick Smith Chemical Co., Columbus, Ohio, 1944. 2. DRABKIN, D. L., J. Biol. Chem. 140, 387-396 (1941). 3. KOLTIIOFF, 1. M., AND SANDELL, E. B., “Textbook of Quantitative Inorganic Analysis,” 3rd cd., pp. 566-567. Macmillan, Sew York, 1957. 4. DELAHAT, P., “Instrumental Analysis,” p. 207. Macmillan, New Pork, 1957. 5. FISCHEH, H., AND ORTH, H., “Die Chemie des Pyrrols.” Akademische Verlagsgesellschaft, Leipzig, Germany, 1934 (a translated version is given in “Organic Syntheses,” Vol. 21).
BIOCHEMISTRY
11,
Determination
159-163
of
(1965)
Iron
in
Heme
Compounds
I. Hemin ALAN From
the Department University
D.
ADLER
AND
of Biology and of Pennsylvania, Received
PHILIP
John Harrison Philadelphia, July
GEORGE Laboratory Pennsylltania
of Chemistry,
7, 1964
Calorimetric determination of the iron in natural products by means of o-phenanthroline has been described by several authors. This literature is reviewed in detail in the comprehensive monograph on phenanthroline analytical methods by Smith and Richter (1) along with most of the pertinent physical and chemical data. The determination of iron specifically in hemin by such methods was first given by Drabkin (2). Drabkin’s method consists in decomposition of the hemin by H,O, in alkaline media, heating to remove the peroxide, acidification and digestion with HCI, buffering to approximately pH 4 with saturated ammonium acetate, reducing from ferric to ferrous iron with ascorbic acid, forming the o-phenanthrolinc complex with excess reagent, and finally calorimetrically determining the optical density at about 500 my (2 cm,kf = 11.05) against a proper blank. This procedure requires several hours for the determination of a single sample. As it is frequently necessary to obtain values for the iron content of iron porphyrins in physical studies of these materials, we have modified the above procedure to provide a somewhat more rapid and convenient determination. METHODS
Deionized water distilled from alkaline permanganate is used throughout this procedure for making up solutions and the final rinsing of all glassware. After initial cleaning the glassware is routinely cleaned with hot tap water and detergent and then rinsed with the distilled deionized water and allowed to dry. The pyridine used should be fresh reagent or spectral grade (or its equivalent). Pyridine exposed to air is observed to yellow very slowly due to the formation of pyridine oxide, etc. The aqueous 2.0 M solution is stable, however, for several months. A hemin sample (10.00 to 15.00 mg) is weighed out on a cleaned and tared glass microscope slide cover slip on a microbalance. The slip is picked up with tweezers and the hemin is washed via a small funnel into 159 01965
by Academic
Press Inc.
160
ADLER
AND
GEORGE
a 25.0-ml volumetric flask with 2.0 M aqueous pyridine solution. The solution is then adjusted to volume with the pyridine solution to form a hemin stock solution. A LOO-ml aliquot of the hemin stock solution is transferred to a 25.0-ml volumetric flask and allowed to react with 0.2 ml of 30% H202 (Merck Superoxal) until gas evolution ceases. The flask is gently agitated and swirled for the 5 min required for the complete decomposition. Then several aliquots of solid recrystallized ascorbic acid sufficient to cover the tip of a microspatula (approximately 0.5 mg/aliquot) are added to the decomposition mixture until no gas evolution occurs upon addition of a further aliquot (generally 3 to 4 aliquots are required). Then 2.0 ml of 0.1% aqueous o-phenanthroline (G. Frederick Smith Chemical Co.) is added and the solution brought up to the mark with distilled deionized water. After allowing 15 min for color development, the optical density is spectrophotometrically determined with a Beckman DU against a blank at 509 mP and the iron content calculated from an c1 Cmmdl= ll.O-the average of the values given by several authors for the tris-Fe%-phenanthroline complex is P C*nmJf= 11.1 at 510 rnp (1). The optical density is rechecked after another 5 min. Occasionally a sample fails to recheck to within 0.001 optical density. Such samples should be discarded and another determination made from the hemin stock solution. The blank comprises all the reagents but the hemin and should be carried through the procedure simultaneously with the hemin sample. However, distilled water blanks may also be used, since the LLdetermination” blank vs. water usually gives readings less than 0.001 optical density. Mohr’s salt, Fe (NH,) 2 (SO,) 2 -6 H,O, was used as the primary standard to check, calibrate, and determine the 8 Cm,M for this procedure. Freshly recrystallized reagent-grade salt was employed in order to avoid the problem of efflorescence (3). Beer’s law was tested for the tris-Fen-ophenanthroline complex under the conditions of this method with Mohr’s salt over the range of optical densities 0.010 to 0.850 and found to be obeyed. This experiment gave the extinction coefficient quoted above. The following changes in procedure were found not to alter significantly the blank, the requisite conditions, the accuracy, or the precision of the method: variation of temperature from 15 to 35”C, variation of pyridine stock solution concentration from 1.0 to 4.0 M, variation of the H,O, employed from 0.1 to 0.4 ml, variation of decomposition time from 2.5 to 10 min, variation of the solid ascorbic acid used from half to treble the usual amount, variation of the o-phenanthroline solution added from 1.5 to 3 ml, variation of color development time from 10 min to 0.5 hr, and variation of wavelength of measurement from 508 to 510 mp. Further,
IRON
IN
HEME
161
COMPOUNDS
the 15-min value of the optical density of both a set of hemin and Mohr’s salt samples were found to be stabIe for five days, after which time they were discarded. Several other oxidants and reductants were also tested for this procedure-H,O, and ascorbic acid remained the most convenient reagents, although Cl, or ClO, and hydroxylamine salts or hydrazine were also found to be suitable. As 25 determinations can be rapidly made upon a single weighing, the precision of the method can be very high. Agreement between such determinations and also between triplicate weighed samples are found to be limited to the precision and accuracy of the Beckman DU spectrophotometer (which is maximally about 0.570). The sample weights employed give optical densities in the range 0.200 to 0.500 where the spectrophotometer gives the best’ compromise between sensitivity and accuracy (4). With practice the entire determination can be carried out in about onehalf hour. The sensitivity of the procedure can be extended by weighing out 0.40 to 0.60 mg of hemin and transferring directly to the decomposition vessel with 1.0 ml of 2.OM aqueous pyridine. It may be further extended by a factor of about 20 by the use of bathophenanthroline and IO-cm light path length cells. The method has been found equally applicable to several other iron porphyrin complescs than protohemin and also to other metalloporphyrins such as the Cu, Co, etc. derivatives. RESULTS
Iron determinations were made upon recrystallized and crude horse blood hemin, prepared according to Fischer (5)) and, also, on four commercial samples purchased from Eastman Kodak, L. Light & Co., Sigma Chemical Co., and California Biochemical Corp. Duplicate weighings were made for each sample, five determinations were performed with each hemin stock solution, and the ten values were averaged. The average % Fe and 70 expected Fe for each sample are given in Table 1. The average deviations for average ‘$% Fe’s are all less than -~0.0170. However, as this represents the precision and accuracy of the DU spectrophotometer TABLE IRON Source
CONTENT
of hemin
OF SIX
sample
Recrystallized (horse blood) Crude (horse blood) Eastman Kodak L. Light & Co. Calif. Biochem. Corp. Sigma Chem. Co.
1
DIFFERENT
HEMIN 70 Fe
8.31 7.20 8.15 8.01 s.01 7.89
S.UVPI,ES % expected
97.1 s4.0 95.5 93.5 93.5 93.1
Fe
162
ADLER
AND
GEORGE
and not of the chemical procedure, its merit as an error estimate is somewhat limited. The theoretical iron content for hemin is 8.5770 by weight. The procedure gave 99.9% of the expected iron content for a single control Mohr’s salt sample run simultaneously with these samples. DISCUSSION
Microscope cover slips and the washing transfer are employed to avoid the problem of hemin samples sticking to surfaces such as weighing paper, etc. As the cover slips weigh only about 100 mg, taring preserves the sensitivity of the microbalance. The presence of pyridine in the decomposition reaction mixture prevents the formation of colloidal iron sols as in the Drabkin procedure. Further, combination of the pyridine and ascorbic acid in the final solution serves to buffer it in the optimum pH range for this determination (1) without addition of any further buffering agents. Though this method gives the same accuracy and precision as the Drabkin method, it is faster and more convenient. Furthermore, the only reagents employed are so low in iron content that in the amounts employed in the determination they contribute no appreciable color to the blank. The 2.0 M pyridine and the 0.1% o-phenanthroline solution are the only required stock solutions. These are readily prepared and both are stable for months (and even for longer under refrigeration and in appropriately sealed containers). No heating steps are required as in previous methods. Finally the method is such that the limiting sources of systemat.ic error are in the initial weighing and in the optical density reading. This is easily further limited to the optical density reading by the proper use of a good microbalance. The interpretation of the wide variation in the results obtained for the six different hemin samples can only be given without speculation when these results are compared with further analytical studies on the C, H, N, 0, and Cl contents of these same samples. As we have performed such further studies, these data will be reviewed and discussed in a separate paper. SUMMARY
An accurate, precise, rapid, and convenient method for the spectrophotometric determination with o-phenanthroline of the iron content of hemin is detailed. The limitations, modifications, and extensions of the method are discussed. The application of the method to six different solid hemin samples is presented to exemplify the utility of the method. This work was supported
ACKNOWLEDGMENT by Public Health Service Grant AM-03187.
IRON
IN
HEME
COMPOUNDS
163
REFERENCES 1. SMITH, G. F. AIVD RICHTER, F. I’., “Phenanthroline and Substituted Phenanthroline Indicators.” G. Frederick Smith Chemical Co., Columbus, Ohio, 1944. 2. DRABKIN, D. L., J. Biol. Chem. 140, 387-396 (1941). 3. KOLTIIOFF, 1. M., AND SANDELL, E. B., “Textbook of Quantitative Inorganic Analysis,” 3rd cd., pp. 566-567. Macmillan, Sew York, 1957. 4. DELAHAT, P., “Instrumental Analysis,” p. 207. Macmillan, New Pork, 1957. 5. FISCHEH, H., AND ORTH, H., “Die Chemie des Pyrrols.” Akademische Verlagsgesellschaft, Leipzig, Germany, 1934 (a translated version is given in “Organic Syntheses,” Vol. 21).