Curing heme pigments

Killday etal. (1988) also provided evidence for internal autoreduction of ferric nitrosyl heme complexes, as previously proposed by Giddings (1977). Heating of chlorohemin( iron-III) dimethyl ester in dimethyl sulfoxide solution with imidazole and NO produced a product with an infrared spectra identical to that of nitrosyl iron(ll) protoporphyrin dimethyl ester prepared by dithionite reduction. Both spectra clearly showed the characteristic nitrosyl stretch at 1663 and 1665 cm. They thus proposed a mechanism for formation of cured meat pigment which includes internal autoreduction of NOMMb via globin imidazole residues. A second mole of nitrite is proposed to bind to the heat-denatured protein, possibly at a charged histidine residue generated in the previous autoreduction step. 

The pink cured meat pigment mononitrosylhemochrome is a complex of nitric oxide (NO), ferrous heme iron, and heat-denatured globin protein (Table F3.2.1). The pink nitrosylheme (NO-heme) moiety may be extracted from the protein in aqueous acetone and quantitated by A540 (see Basic Protocol 1). The percent nitrosylation may be determined from measurement of ppm NO-heme relative to ppm total acid hematin (hemin) extracted in acidified acetone (see Basic Protocol 2), since NO-heme is completely oxidized to hemin in acid solution (i.e., 1 ppm NO-heme = 1 ppm hemin). 

The measurement of cured meat pigment concentration is based on the A540 of the nitrosyliron(II)protoporphyrin group (also known as nitrosylheme or NO-heme mol. wt. 646) in an extraction solution of 80% (final) acetone in water, taking into consideration the 70% water content of the meat sample. Hornsey (1956) established that only the pink NO-heme was extracted in 80% acetone. Heme groups from fresh meat pigments (Table F3.2.1) are not extractable in 80% acetone. However, upon acidification with hydrochloric acid, NO-heme in 80% acetone was completely oxidized to hemin. Thus, NO-heme concentrations could be expressed in equivalent ppm hemin. 

Nitrosylheme, also known as NO-heme or nitrosyliron(II) protoporphyrin, is readily extracted from the cured meat pigment mononi-trosylhemochrome (Killday et al., 1988) into an aqueous acetone solution for quantitation by spectroscopy. Hornsey (1956) established the following important points for accurate NO-heme quantitation. 

The Hornsey (1956) procedure and its modifications have received widespread acceptance as relatively rapid measures of the adequacy of cure development in processed meats. The Hornsey procedure is also an accurate method for nutritional assessment of heme and heme iron content of meats (Carpenter and Clark, 1995), where ppm heme iron = ppm total heme/11.7. However, one caveat should be noted. The total heme pigment measurement is higher in cured meats than in similar uncured samples. Roasted turkey breast meat, for example, was reported by Ahn and Maurer (1989a) to have 23,26,34, and 34 ppm total pigment in samples formulated with 0, 1, 10, and 50 ppm nitrite, respectively. This effect should be considered to avoid overestimation of the heme iron content of cured meats. 

About 80% to 90% conversion of heme pigments to nitrosylhemochrome is desirable for cured meats (Pearson and Tauber, 1984). However, beef pastrami with typical cured color may have as low as 62% conversion (94 ppm NO-heme out of 153 ppm total heme Cornforth et al., 1998). 

This paper provides the basic information for spec-trophotometric determination of nitrosyl and total heme pigment levels in cured meats. 

This paper is an adaptation of the Hornsey (1956) method for measurement of nitrosyl and total heme pigments in cured meats, using 2 g meat samples rather than 10 g samples. Thus, less reagent is needed and more samples may be analyzed at the same time. 

Killdav, K. B. Tempcsta, M. S. Bailey, M. E. Isolation and Characterization of Heme Pigments from Cured Meat. Presented at the 20th Midwest Regional Meeting of the American Chemical Society, Carbondale, IL, 1985. 

Sebranek (1988) has reviewed the effects of heat on denaturation of the proteins. Dehydration by heat denatures the muscle proteins, particularly the sarcoplasmic proteins. This induces a rather dramatic change in meat color. The heme pigments, which provide most of the color of fresh meat, serve as a general indication of doneness or temperature history. In the case of cured products, heme pigments react to form nitric oxide hemochro-mogen, which contributes the characteristic pink cured meat color (Pearson and Tauber, 1984). 

Greenwood, D. A., Lewis, W. L., Urbain, W. M., and Jensen, L. B. 1940. The heme pigments of cured meats. IV. Role of sugars in color of cured meats. Food Research 6, 625. 

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