Quantitative determination of myoglobin and haemoglobin in beef by high-performance liquid chromatography

Meat Science 28 (1990) 313-320

Quantitative Determination of Myoglobin and Haemoglobin in Beef by High-Performance Liquid Chromatography

Inger M. Oellingrath,* Arve lversenl: & Grete Skrede§ MATFORSK, Norwegian Food Research Institute, Oslovn 1, 1430 As, Norway (Received 28 October 1989; revised version received 20 December 1989; accepted 5 January 1990)

A BSTRA CT Three different HPLC columns were tested for their suitability for the analysis of myoglobin and haemoglobin in beef Complete separation of the two proteins was obtained with an anion exchange column and a column based on hydrophobic interaction. With the latter column a recovery close to 100% was obtained for both proteins when they were added to meat extracts. For both proteins, the standard error of the mean for repeated analyses of ground beef was less than 2% of the detected amount. The method is considered as suited for quantitative routine analysis of myoglobin and haemoglobin in beef and beef products.

INTRODUCTION The major pigments o f meat are the muscle protein myoglobin and the blood protein haemoglobin. The haemoglobin content depends on the extent o f the vascular bed in the muscle and the bleeding of the carcass. Generally only * Present address: Norwegian Dairies, PO Box 9051, Vaterland, N-0134 Oslo 1, Norway. Present address: Troll Salmon A/S, PO Box 40, N-1622 Engalsvik, Norway. §To whom cort:espondenceshould be addressed. 313 Meat Science 0309-1740/90/$03"50 © 1990 ElsevierScience Publishers Ltd, England. Printed in Great Britain

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a small part (approximately 10%) of the total pigment of meat is haemoglobin (Warriss & Rhodes, 1977). Quantitative determination of the two heme proteins in raw materials is essential in colour optimalization of meat products (Oellingrath & Slinde, 1985). Myoglobin and haemoglobin may be analysed by gel filtration techniques as described by Warriss (1976). A more time-saving technique is high performance liquid chromatography (HPLC). The method has been used for the determination ofmyoglobins (Powell et al., 1984) and for the separation of various variants of haemoglobin (Robinet et al., 1984). Ghrist et al. (1987) reported different chromatographic properties of myoglobin and haemoglobin when analysed by HPLC. The small amount of haemoglobin compared with the amount of myoglobin has been a problem in qualitative and quantitative determination of the two proteins in meat extracts. Chromatographic separation of myoglobin and haemoglobin in meat extracts by HPLC was obtained by Oellingrath & Slinde (1985). However, the analytical conditions were considered as unsuited for routine analysis since the two proteins eluted as one peak after prolonged use of the column. The purpose of the present study was to optimize the analytical conditions for qualitative and quantitative determination of myoglobin and haemoglobin in beef extracts by HPLC and to develop a method suitable for routine measurements of the two heme proteins in beef.

MATERIALS AND METHODS Standard solutions

Commercial horse skeletal myoglobin and bovine haemoglobin (Sigma Chemical Company, St Louis, MO) were purified to exclude polymerized material as described previously (Oellingrath & Slinde, 1985). Once purified, the standard solutions were stable for several weeks when kept frozen in their cyano ferric form. The purified standards were diluted (1:50v/v) in buffer (0"IM sodium phosphate, 0"01M KCN, pH 7.0). The amounts of myoglobin and haemoglobin in solutions were determined as the cyano ferric form of the heme proteins by scanning the Soret region, 400-450nm (Oellingrath & Slinde, 1985). The pigment concentrations were calculated using the millimolar extinction coefficient at 422nm (EmM= 116.0) for horse skeletal cyanomyoglobin and at 419nm (EmM= 124"0) for horse cyanohaemoglobin (Antonini & Brunori, 1971). Molecular weights of 18500 and 64500 were used for myoglobin and haemoglobin, respectively.

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315

Chromatographic systems A Spectra Physics model 748 HPLC equipped with a Biotronik BT 3030 UV-detector (Biotronik Wissenschaftliche Ger/ite GmbH, West Germany) and a Spectra Physics 4270 integrator was used throughout the experiments. Three different columns were tested at the conditions suggested by the producer (Bio-Rad Laboratories, CA). The analytical conditions were optimized by adjusting the pH of the mobile phases and the slope of the elution gradient in preliminary experiments.

Gelfiltration column: A Bio-Sil TSK-SW (600 x 7-5 mm) molecular sieve column with a Bio-Sil TSK guard column (75 x 7.5 mm) was used. The column was eluted with 0"05M sodium phosphate, 0"01M K C N and 0"IM Na2SO4, pH 7.22 (Oellingrath & Slinde, 1985). Ion exchange column: A D A E D Microanalyzer MA 7P Cartridge (30 x 4-6 mm) anion exchange column was eluted with 20 mM trishydroxymethylaminomethane, pH8.5 (eluent A) and 20mM trishydroxymethylaminomethane, 0"5M NaC1, pH 8-5 (eluent B). The linear gradient was 0-100% B in A during 5 min. Hydrophobic interaction column: A Bio-Gel TSK Phenyl-5-PW (75 x 7-5 ram) column was used. The column was operated with l'7M (NH,~)2SO ~, 0-1M sodium phosphate, pH7"0 (eluent A) and 0"IM sodium phosphate buffer, pH 7.0 (eluent B). The linear gradient was 0-100% B in A during 15 rain. Samples of 10/A were injected onto the column. With all systems the flow rate was 1 ml/min. Absorbances were read at 420 nm. The detector response was linear within the concentration range tested (0-1-1.2 mg/ml) and peak areas were used for quantitative calculations.

Sample preparation To 10g of minced beef (15% fat) 30ml extraction buffer was added. The extraction buffers were the mobile phase for the gel filtration column, eluent B with the addition of a few mg K3Fe(CN)6 for the anion exchange column, and eluent B for the hydrophobic interaction column. The samples were homogenized in a Colworth Stomacher 400 for 1 min and centrifuged ( 106 g)av at 2°C for 30 min (Oellingrath & Slinde, 1985). The supernatant was filtered through microfilters (0-45/~m). A few mg of K C N was added to keep the heine proteins in their stable cyano ferric form (Warriss, 1976). The solutions were injected onto the chromatographic columns.

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Known amounts of myoglobin and haemoglobin standards were added to minced beef extracts to test the analytical recovery of the proteins. RESULTS

Separation of myoglobin and haemoglobin by HPLC Chromatograms of myoglobin and haemoglobin standards analysed with the three different chromatographic systems are shown in Figs 1 and 2. With the gel filtration column (Fig. I(A)) haemoglobin had a shorter retention time than myoglobin. With the anion exchange column (Fig. I(B)) and the column based on hydrophobic interaction (Fig. 2(A)) the order of elution was reversed. The anion exchange column and the hydrophobic interaction column both separated myoglobin and haemoglobin completely. The time of analysis was shorter with the anion exchange column than with the hydrophobic interaction column, the retention time for haemoglobin being 6 and 15 min, respectively. B

A

Mb

Mb

Fig. 1. (A) Best achieved separation of standard solutions of haemoglobin (Hb), retention time = 20-3 min, and myoglobin (Mb), retention time = 24-0 min, on Bio-Sil TSK-SW gel filtration column. (B) Separation of standard solutions of haemoglobin (Hb), retention time = 4-1 min, and myoglobin ~(Mb), retention time=0-3min, on DAED Microanalyzer MA 7P Cartridge anion exchange column. Analytical conditions as described in the text.

I

I

0

5

I

I

I

l

I

10 15 20 25 30

0246810

RETENTION TIME (min)

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317

B

A I

4 Mb

Hb

Fig. 2. Separation of haemoglobin (Hb), retention time = 5 10 15 20 5 10 15 20 Retention time (min.)

14.0, and of myoglobin (Mb), retention time = 7"8 min, on Bio-Gel TSK Phenyl-5-PW hydrophobic interaction column. (A) Standard solutions; (B) ground beef extract. Analytical conditions as described in the text.

A m o n g the columns tested, the column based on hydrophobic interaction (Fig. 2(A)) produced the best separation of the myoglobin and haemoglobin standards. Separations were obtained in both standard solutions (Fig. 2(A)) and in meat extracts (Fig. 2(B)) and the column was chosen for further studies of heme protein analysis in meat extracts.

Recovery of myoglobin and haemoglobin in meat extracts Recovery studies of haemoglobin and myoglobin were performed by adding heme protein standards to aliquots of meat extracts. The amount of myoglobin and haemoglobin added corresponded to about 1 mg/g meat for each. The myoglobin standard caused a split in the myoglobin peak when the standard was analysed along with the meat extracts. The horse myoglobin of the standard eluted about 0.65 min after the beefmyoglobin of the meat extract. The two myoglobin peaks were integrated separately. The recoveries of the heme proteins, calculated as the percentage of the a m o u n t added, are presented in Table 1. Three series of experiments were performed, each consisting of four analyses. For both myoglobin and

lnger M. Oellingrath, Arve lversen, Grete Skrede

318

TABLE 1 Percent Recovery (%) and Standard Deviation (s) from three Experiments with Myoglobin and Haemoglobin added to Minced Beef Extracts

Experimental series

Myoglobin

Haemoglobin

%

s

%

s

1 2 3

97-3 113"3 87"3

2'5 11"7 2"1

100"0 107'8 91"0

18"0 5"4 1"4

Average SEM

99-3 10"7

5"4 --

99.6 6'9

8-3 --

haemoglobin, the recovery ranged from about 90% to about 110%. Average recoveries from the three experiments were close to 100%. However, the standard errors of the means (SEM) indicated an overall variation of about 10% in the recovery of the heme proteins when added to meat extracts.

Determination of myoglobin and haemoglobin in minced beef The ability of the chromatographic method to determine myoglobin and haemoglobin concentrations of minced beef samples was examined by extracting heine proteins in samples of the same batch of meat. Each extract was subjected to repeated injections onto the HPLC column. The results are presented in Table 2. The standard deviation by repeated injections of each extraction was less than 1"2% of the concentration detected for myoglobin and less than 1-8% TABLE 2 Mean Value (x) and Standard Deviation (s) of Myoglobin and Haemoglobin Concentrations determined by Repeated Injections (n) of Extracts from four Samples taken from one Batch of Minced Beef

Sample

A B C D Average SEM

n

10 6 5 4

Myoglobin (mg/g)

Haemoglobin (mg/g)

X"

S

X

S

6"56 7"20 7"04 6-80

0-08 0-05 0"06 0"08

0"620 0"652 0"680 0"652

0-011 0-003 0"009 0"009

6-90 0-24

0-07 --

0.651 0-021

0.008 --

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for haemoglobin. In the four replicate extractions the standard error of the mean (SEM) amounted to less than 3.5% of the average concentration for both heme proteins examined. Haemoglobin represented 8-6% of the total amount of myoglobin and haemoglobin in the minced beef extracts.

DISCUSSION As reported previously (Oellingrath & Slinde, 1985), the separation of myoglobin and haemoglobin from minced beef extracts was not complete even after the pH and the ionic strength of the mobile phase had been varied when the gel filtration column was used. The gel filtration column of the present experiment was considered as unsuited for the analysis of a mixture of myoglobin and haemoglobin. Differences in retention times by HPLC chromatography of myoglobin and haemoglobin have been reported by Ghrist et al. (1987) when they used a zirconia-stabilized silica particle packing stabilized with diol bonding. The proteins were analysed under both denatured and non-denatured conditions. No information about whether the two components were completely separated was given. In the present experiments both the ion exchange column and the column based on hydrophobic interaction separated myoglobin and haemoglobin completely. The analytical time was shorter for the ion exchange column than for the hydrophobic interaction column. When the aim of the analysis is a separation and a quantification of myoglobin and haemoglobin, the heme group of the protein should be converted to one state of oxidation prior to analysis. In the present study this was done by adding a small amount of K3Fe(CN)6 or K C N to the proteins prior to analysis. The ferric form of the heme iron was maintained by the further addition of K C N to the solutions (Oellingrath & Slinde, 1985). Preliminary studies with the ion exchange column indicated high sensitivity to small differences in ionic properties of the protein for this column. This possibly makes the column suitable for separation of different red-ox states of myoglobin and haemoglobin. Various red-ox states of the heme group would produce small differences in the charge of the myoglobin and haemoglobin molecules (Antonini & Brunori, 1971). High ionic strength of the mobile phase, as often used in reversed phase chromatography, causes proteins to elute in denatured form. The eluting conditions of the hydrophobic interaction column used in the present experiments, however, did not cause denaturing of the myoglobin and the haemoglobin molecules as the relatively weak hydrophobic interaction

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caused by the constituents of the column matrix requires only nondenaturing salt gradients for elution. This makes it possible to perform a selective measurement of the intact heine proteins. By eluting undenatured myoglobin and haemoglobin the light-absorbing heme group can be used for their selective detection in the presence of non-haem proteins. When chromatographed on the hydrophobic interaction column, beef myoglobin of the samples was separated from the horse myoglobin used as standard. This is likely to be due to species differences of myoglobin. By using an ionic exchange HPLC column, Powell et aL (1984) reported that, out of two peaks for human myoglobin,, one peak eluted separated from horse myoglobin. Further, differences in myoglobin between beef and horse have been utilized for species identification by isoelectric focusing (Hofman & Blfichel, 1986). The separated myoglobin peaks of the present method may indicate a possibility for developing a method for species identification based on HPLC. The contents of myoglobin (6.9mg/g) and haemoglobin (0"65mg/g) detected in the minced beef samples were in the range of values reported previously (Warriss, 1976; Warriss & Rhodes, 1977). Thus, the method appeared to be reproducible, sensitive and rapid enough to have a potential as a routine method for the analysis of myoglobin and haemoglobin in meat samples.

ACKNOWLEDGEMENT The technical assistance of Tone Bergersen is gratefully acknowledged. The authors thank Bio-Test A/S, Norway, for providing the HPLC columns free of charge.

REFERENCES Antonini, E. & Brunori, M. (1971). Hemoglobin and Myoglobin in their Reactions with Ligands. North-Holland Publishing Company, Amsterdam. Ghrist, B. F. D., Stadalius, M. A. & Snyder, L. R. (1987). J. Chrom., 387, 1. Hofman, K. & Bliichel, E. (1986). Fleischwirtsch., 66, 916. Oellingrath, I. M. & Slinde, E. (1985). J. Food Sci., 50(6), 1551. Powell, S. C., Friedlander, E. R. & Shihabi, Z-K. (1984). J. Chrom., 317, 87. Robinet, D., Sarmini, H., Lesure, J. &Funes, A. (1984). J. Chrom., 297, 333. Warriss, P. (1976). Anal Biochem., 72, 104. Warriss, P. D. & Rhodes, D. N. (1977). J. Sci. Food Agric., 28, 931.