Isolation and Purification of Turkey Heme Proteins

Abstract

Turkey heme proteins, myoglobin, hemoglobin and cytochrome c were isolated and their interactions were studied. This report describes procedures for the chromatography of myoglobins on CM cellulose, DEAE cellulose and Sephadex columns. DEAE cellulose was used to separate hemoglobin and cytochrome c.Turkey metmyoglobin was shown to display a heterogeneity as demonstrated in CM cellulose column chromatography, polyacrylamide gel electrophoresis, and isoelectric fractionation. Three electrophoretically distinct myoglobins were observed in gel electrophoresis, and seven myoglobins were separated by isoelectric fractionation. The major myoglobin fraction was found to have an isoelectric point of 7.99.Turkey myoglobin was similar to chicken myoglobin, but different from mammalian myoglobin in amino acid composition and molecular weight. Molecular weight of turkey myoglobin was determined to be 18,199.The autoxidation rate of turkey myoglobin exhibited a 6-fold slower rate in the presence of cytochrome c. The cytochrome c reduction of metmyoglobin to reduced myoglobin was the probable mechanism.The heat denaturation of turkey myoglobin is quite similar to myoglobins from other species (1/2 D = 78.0° C). The denaturation temperature increased by approximately 1° C. as the other heme proteins were added to myoglobin, indicating a protein-protein interaction, thus allowing myoglobin to resist heat denaturation. The heat denaturation study revealed that cytochrome c resists denaturation at 105° C.

Mass spectrometric characterization and thermostability of turkey myoglobin

“…The molecular mass of purified turkey Mb determined by MALDI-TOF MS was 17,295 Da, which was greater than that of beef Mb (16,949 Da), and suggests a different primary structure. Our results on turkey Mb's molecular mass are different from those of Cornish and Froning (1974) who estimated its molecular mass as 18,199 Da, based on amino acid composition analyses. This variation is attributed to the use of high throughput and accurate MS tools used in our study that were not previously available.…”

“…Frozen turkey hearts were procured from a local purveyor. Turkey hearts were thawed overnight, trimmed of fat, homogenized in 20 mmol/L ammonium bicarbonate buffer (pH 9.0, 4 C) at 1:1 ratio, and centrifuged at 5000 Â g for 10 min at 4 C. The reported isoelectric pH (pI) of turkey Mb (pI ¼ 7.99; Cornish & Froning, 1974) is greater than that of beef Mb (pI ¼ 7.1; Satterlee & Snyder, 1969). Therefore, after trial-and-error, a homogenization buffer with a high pH (9.0) was chosen for Mb extraction rather than using Tris-EDTA buffer with pH 8.0 described by Faustman and Phillips (2001).…”

“…The molecular weight of red meat (beef, pork, lamb, water buffalo, horse, yak, and red deer) myoglobins is 17 kDa (www.expasy.org). On the other hand, the estimated molecular weight of turkey Mb, based on amino acid composition analyses, is 18.2 kDa (Cornish & Froning, 1974). However, in the preliminary work from our lab, turkey Mb appeared as a 17 kDa band in Coomassie blue-stained sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gels.…”

“…In contrast, Nam and Ahn (2002) reported formation of carboxymyoglobin as the reason for PCD in irradiated turkey meat. Interestingly, the presence of other heme proteins, such as cytochrome C, increased the resistance of turkey Mb against heat-induced denaturation, possibly through proteinprotein interactions (Cornish & Froning, 1974). While investigating species-specific factors contributing to PCD, Trout (1989) that at pH inherent to respective meats, heat-induced Mb denaturation was lower in turkey (pH 6.0) compared to beef and pork (pH 5.5).…”

Primary structure of turkey myoglobin

“…Interestingly, turkey myoglobin shares 100% sequence similarity with chicken myoglobin. Previously, Cornish and Froning (1974) suggested high similarity in amino acid residues in turkey and chicken myoglobins, based Table 1 Sequences of peptides generated after enzymatic digestion of turkey myoglobin with trypsin and aspartic acid endopeptidase (Asp-N).…”