low the up-grading of a good deal of the present 

 pack. The natural pink color of canned tuna 

 is due to substances known as denatured hemo- 

 chromes and hemichromes which are formed 

 during the precooking stage from the heme 

 pigments of the tuna (Brown and Tappel, 

 1957). Two heme pigments occur naturally 

 in tuna — myoglobin found in the muscle and 

 hemoglobin in the blood. There is usually con- 

 siderably more myoglobin in the "white" dorsal 

 muscle of the tuna used for canning for human 

 consumption. Both these compounds are nor- 

 mally deep red proteins which are concerned 

 with oxygen transport in the living fish. (The 

 role of myoglobin in oxygen transport has not 

 been clearly elucidated.) They are closely sim- 

 ilar to the red heme compounds which give the 

 color of meat and perform similar functions in 

 land animals. The amount of these compounds 

 in tuna is believed to be the major factor con- 

 trolling the intensity of the tuna color that is 

 important commercially. Myoglobin comprises 

 about 85% of the total heme pigments in the 

 white muscle. Thus, albacore, the lightest 

 colored of the tunas, contains an average of 

 0.15% of myoglobin in the white muscle; yel- 

 lowfin, which is darker, 0.22%; and skipjack, 

 usually considered the darkest of the tunas, 

 0.4%. The red meat of tuna (the highly vas- 

 cular, hemoglobin-rich dorsal muscles canned 

 for pet food) with its dark red-brown color 

 contains 3% or more. The amounts of pig- 

 ments present within each species also show 

 some variation. It is not likely that much can 

 be done to change the amount of these pig- 

 ments present in tuna, but it seems possible 

 that the condition of the pigment myoglobin, 

 which is present in the largest amount, may be 

 more important to the color in any one species. 

 For instance, large yellowfin are generally 

 darker than smaller yellowfin on cooking and 

 canning, but the few figures available on dif- 

 ferent sized yellowfin seems to indicate that 

 large yellowfin contain no more heme pigments 

 than the small (Crawford et al., 1969) . In this 

 case then, the darker color may be altered by 

 changing the storage condition in a way which 

 favors a better color. Therefore, experiments 

 were conducted on the occurrence of heme pig- 

 ments in tuna, their state of oxidation and 

 other changes to determine how they aflfect the 

 color of the canned product. Figure 3 summa- 



rizes the changes in myoglobin from the time 

 the tuna is caught and processed. 



Derivatives of hemoglobin and myoglobin 

 are formed in the precooking and canning pro- 

 cess ; a knowledge of the changes in which they 

 are involved is important in understanding and 

 influencing color development in the end prod- 

 uct. Earlier work by Brown et al. (1958) and 

 other investigators had shown that oxidation 

 plays an important part in cooked color, the 

 oxidized or met forms being brown as com- 

 pared with the usual pink color of the oxy 

 form. Also the phenomenon of "greening" un- 

 doubtedly involves the heme pigments. Gros- 

 jean et al. (1969) and Koizumi and Matsuura 

 (1967) reported that "greening" resulted from 

 the interaction of free cysteine, myoglobin, and 

 TMAO. Accordingly these studies have been 

 concerned with the stability to autoxidation of 

 the heme pigments and their derivatives under 

 the differing conditions of pH and temperature 

 likely to occur in tunas being handled through 

 regular commercial channels. The first step 

 was the predation of pure myoglobins by am- 

 monium sulfate fractionation and DEAE-cel- 

 lulose chromatography from tuna muscle. To 

 study oxidation, the purified yellowfin and skip- 

 jack metmyoglobins and hemoglobins were dis- 

 solved in a citric acid-phosphate buffer and re- 

 ducted to the oxy form found in the fish. These 

 solutions at various pHs from 5.7 to 6.3 were 

 then exposed to oxygen at differing temper- 

 atures and the course of autoxidation followed 

 visually by color change and by spectrophoto- 

 metric measurements of the 580 m^a peak which 

 is characteristic of the oxy form. The results 

 showed that the heme pigments all oxidize more 

 rapidly at lower pH, and the rates of oxida- 

 tion are directly proportional to the hydrogen 

 ion concentration. A reaction mechanism pos- 

 tulated was: 



K, 

 H+ + MbOa (Hmb)^ + O2 (rapid 



K2 equilibrium) 



K3 

 '''^02 + (HMb)^ > MetMb + 1/0 H2O 



Our notable point was that while the rates 

 of oxidation decreased with temperature under 

 the same conditions down to freezing, the re- 

 verse occurred with the frozen solutions. Thus 



