THE CHEMISTRY OF HÆOGLOBIN AND MYOGLOBIN IN RELATION TO THE COLOR OF MEAT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By HOWARD NED DRAUDT, B.Sc., M.Sc. The Ohio State University 1955 Approved by: Adviser The Department of Agricultural Biochemistry TABLE OF CONTENTS Page INTRODUCTION ........................................... 1 LITERATURE SURVEY ....................................... 3 Myoglobin and Hemoglobin.... .................... 3 Methemoglobin or Metmyoglobin Formation ......... 7 The Action of Nitrites on Hemoglobin and Myoglobin ... 11 The Effect of pH on Curing ..... 18 The Effect of Reducing Agents in Meat Curing ....... 19 Heating and Hemochrome Formation... ................ 21 Color Loss in Cured Products ..... 2k EXPERIMENTAL PROCEDURE .................................. 27 Obj ect of the Investigation ......... 27 The Effect of Heating on Color Fixation and Color Stability.......... 29 Isolation of Metmj'-oglobin ........................ 37 Qualitative Experiments on the Effect of Possible Meat Components on Discoloration ...................... 39 Spectroscopy of the Pigment ...... 54 Manometric Experiments ....... 6l Preparation of Samples ........ 62 Warburg Experiments with Heat Fractionated Pigment ... 65 Gas Uptake in the Presence of Sodium Pyruvate ...... 70 Results of Warburg Experiments with Purified Pigment in which Nitrate and Nitrite were not Determined ..... 72 ii Page The Effect of Oleic Acid on Oxygen Uptake ........... 74 Nitrate and Nitrite Production . «................. 80 Results of Warburg Experiments in which Nitrate and Nitrite were Determined .......................... 83 DISCUSSION............................................. 91 SUMMARY AMD CONCLUSIONS ............................. 95 BIBLIOGRAPHY ........................................... 97 AUTOBIOGRAPHY.................... .................... 10$ 1 1 1 List of Tables Table Title Page 1 Visual Color Scores for Cured Meat Irradiated in the Presence and Absence of Potassium Ferricyanide . 34 2 Visual Color Scores for Cured Meat Irradiated in the Presence and Absence of Potassium Ferricyanide Experiment 2 ........................... 35 3 Color Score After Shaking Pigment with Fat Derivatives ............................. « ... 45 4 The Effect of the Acid Group on Visually Scored Color Loss for Cured Meat Pigment ......... 47 5 Color Scores for Heat Denatured Globin Myohemichrome Irradiated in the Presence of Surface Active Agents and Acetone .................... 51 6 The Effect of Fatty Materials on Discoloration of Denatured Globin Myohemichrome in the Light and in the Dark ................................. 53 7 The Effect of Fatty Materials on Denatured Globin Myohemichrcane Discoloration ...................... 54 8 The Effect of Oxygen Pressure on Rate of Oxygen Uptake .......................... 67 9 Oxidation of Denatured Globin Nitric Oxide Myo- honochrcme in the Presence of Sodium Pyruvate... 71 10 The Effect of Oleic Acid on the Oxygen Uptake of the Heat Denatured Pigment ................... 76 11 The Effect of Oleic Acid on Oxygen Uptake..... 78 12 Nitrate and Nitrite Produced and Oxygen Taken Up J.n the Presence of Light with KOH in the Wells ....... 84 13 Nitrate and Nitrite Produced and Oxygen Taken Up in the Dark with KOH in the Wells .......... 85 14 Nitrate and Nitrite Produced and Oxygen Taken Up in the Presence of Light without KOH in the Wells ..... 89 IV List of Illustrations Figure Title Page 1 Absorbance Versus Wavelength for Denatured Globin I'lyohemichrome and for Denatured Globin Nitric Oxide %rohemochrome After Irradiation ............ 57 2 Absorbance Versus Wavelength for Pigment Samples Prepared as for the Manometric Experiments ........ 58 3 Absorbance Versus Wavelength for Pigment Samples Prepared as for the Manometric Experiments (Curve 2) ...................................... 59 4 Rate of Net Gas Uptake for Partially Purified Denatured Globin Nitric Oxide I^oheraochrome ....... 68 5 Rate of Oxygen Uptake for Partially Purified Denatured Globin Nitric Oxide Myohemochrome in Pure Oxygen and in Air ......................... 69 6 Rate of Net Gas Uptake for Denatured Globin Nitric Oxide Myohemochrome .......................... 73 7 Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Myohenochrome in the Presence of Methyl Oleate in Pure Oxygen ......................... 77 8 Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Myohemochrome and Denatured Globin Myo­ hemichrome in the Light and in the D a r k ........... 79 9 Visually Observed Rate of Color Loss in the Light (a ) and in the Dark (B) ................. 86 10 Rate of Color Loss and Rate of Oxygen Uptake of Denatured Globin Nitric Oxide Hemochrome in the Light in Air ................................... 87 11 Rate of Nitrate and Nitrite Production from Denatured Globin Nitric Oxide >îyohemochrome ..... 88 ACKKOV.JLEDGEMSNTS The author wishes to express his appreciation to Professor F. E. Deatherage, under whose direction this work was carried out, for his helpful suggestions. The author is indebted to The Ohio State University for the facilities provided. The financial help provided the author by Kingan Incorporated made it possible for the author to carry out this work and is gratefully acknowledged. Helpful suggestions were provided by Dr. R. E, Morse and Dr. M. C. Brockmann of Kingan Incorporated as well as by Professor J. E. Varner of the Department of Agricultui'al Biochemistry of The Ohio State University. VI INTRODUCTION In the presence of oxygen and light, the nitric oxide deriva­ tives of cured meat, either nitric oxide myoglobin or denatured globin nitric oxide myohemochrome, are oxidized to the ferric form with the production of undesirable color changes from red to brown. This oxidative process has received remarkably little study considering the economic loss to the meat packing industry and eventually to the consumer. This process has become especially important since the advent of self service merchandising and pre­ packaging of cured meat products. Examples of this oxidation may be observed at almost any meat counter and may include dark brown weiners, bologna that has lost its fresh appearance and similar products that have lost their appeal to the potential consumer due to discoloration. This undesirable color change involves the nitric oxide derivative of either myoglobin or denatured myoglobin. These derivatives are formed by curing, a process involving nitrite salts either added as such or formed by bacterial reduction of nitrate salts and reducing activity either associated with the enzyme systems of the tissue or with reducing groups developed during heating. The art of curing is an ancient one but has still not yielded ccanpletely to scientific study. An understanding of curing necessarily had to come after some knowledge of hemoglobin was gained. The period from about 186$ to 19OO marked an active period in the study of hemoglobin (1). The first comprehensive work on the nature of the cured meat pigment and a model, nitric oxide hemoglobin came in 1901. At that time, Haldane (2) observed and explained many of 1 2 the facts now known about meat curing. While curing is concerned with formation of the nitric oxide derivative of the heme pipients, the work to be discussed here is largely concerned with the destruction of these derivatives. The chemistry of curing is thus of interest, inasmuch as it gives an insight into the possible chemistry of cured meat discoloration and to the related question of preservation of color. Lack of published work on the chemical reactions involved in fading has been a serious handicap to technologists who wish to inçrove the color stability of cured meat products. Lack of information in this field is perhaps due more to a lack of adequate experimental methods suitable for attacking this problem than to any other cause. Meat is a complex biochemical system and the molar quantity of myoglobin is very low. Also, in practice many meat products are heated during their manufacture in which case the pigment becomes water insoluble. It was thus desirable to study the chemical nature of purified heat denatured pigment with the objective of gaining some insight into the reactions that may be involved in discoloration. LITERATÜHE SURVEY Myoglobin and Hemoglobin Although the hulk of the scientific work done with pm-ified pigments has been carried out with hemoglobin, it would appear to be at best a good model for the pigment, myoglobin or muscle hemoglobin. Most of the blood is removed from the carcass at the time of slaughter and thus most of the pigment of meat is myoglobin. Hemoglobin is perhaps one of the most thoroughly studied of all organic biological substances, yet myoglobin had received very little attention until Theorell (3) prepared it in crystalline form in 1932. Since the literature on hemoglobin is so voluminous and since it is covered comprehensively in two recent books {k) (5), it will be covered only briefly here. Myoglobin has a m.olecul.ar weight of approximately 17,000 and one hane group whereas hanoglobin has a molecular weight of 68,000 with four heme groups. Although they are similar in many of their chemical properties, such as the ability to undergo reversible oxygenation, the myoglobin molecule cannot be considered to be simply one quarter of a hemoglobin molecule. In the live animal much more hemoglobin than myoglobin is present. Drabkin reported that an entire