For observing interspecific relations between fishes living together around the outside of the Zostera area in Matsushima Bay, Miyagi Prefecture, composition of food was investigated on each of the eight fish species. Basing on the interrelation of food compositions, a trophic order within this fish group was presented just as in the series of the fish from the top to the bottom shown in Table 1. Sebastes inermis, “Mebaru”, grown and emigrated out of the Zostera area, is limited more strictly its feeding activities by many highly ranked fish species in this community than while it lived in the Zostera area. Among them, Sebastes oblongus and Sebastes schlegeli, predate the young of Sebastes inermis, predominated inside of the Zostera area. Among many problems involved in interspecific relations between fishes, relations between “Mebaru” and its food organisms, previously reported qualitatively, were analysed quantitatively by feeding experiments and the results will be reported in the third of this series.

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In this experiment, it was tried to fractionate and characterize the browning products in glucose-lysine system.
Colored products and fluorescent products were cationic. Brown pigments were combined irreversibly with Dowex 50, and they showed fluorescence.
Colorless fluorescent substances having ultraviolet absorption at 250 mμ were able to be eluted from Dowex 50 with dilute HCl.
On paper chromatograms, 4-5 colored, 5-6 fluorescent, and 5-6 ninhydrin reactive spots were detected. Spots corresponding to lysine, glucose, and glucosamine displayed strong fluorescence. A fluorescent and ninhydrin reactive spot with smaller R_f value than lysine was detected. Brown pigment which could not be developed showed considerable fluorescence.
All of fractions by paper chromatography showed maximum fluorescence at 450 mμ, and maximum absorption at 265-275 mμ.

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To study the chemical and physical change of liver oil during a process of oxidation, sample oil was oxidized in air at 45°C. with sea-sand, extracted at certain intervals with various solvents as illustrated in the Tables 1-7. As in shown in the tables, in the earlier stage of oxidation, the properties of oils differ little depending on the solvent. In the advanced stage of oxidation, however, remarkable difference was observed in properties of oil depending on the solvent used for the extraction.
Another experiment was done on the samples oxidized for 4 and 7 days, to observe the effect of heat on the nature of the oil during the evaporation of solvent. Considerable differences were found between a oil extracted at room temperature and the one refluxed with sol-vent at boiling temperature for four hours. As in shown in Table 8, increase in acid value, saponification value, oxidized acid and refluctive index, and decrease in iodine value and peroxide value were confirmed to occur by heat treatment.
It is concluded that to obtain true data on an oxydized oil the solvent should be removed under vacuum at room temperature.

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The present investigations were undertaken to know the effect of loading on fish with special reference to the change of phosphorus compounds in the muscle. Gibels were burdened with lead weighing 2 to 25 percent of the body weight.
The results are summarized as follows;
(1) 7'P and CP in the muscle were affected by the load in the range of 10 to 20 percent of the body weight. The lowest value of them was obtained with the load of 15 percent as seen in Fig. 1.
(2) The load of 15 percent produced also a marked effect on the post-mortem change of the above two compounds. As seen in Fig. 2, those decreased more rapidly than in the control and the fish burdened with the load of 5 percent.
(3) It comes to a conclusion that phosphorus compounds in the muscle of gibel were affected before and after the death by the physiological work or fatigue caused by the load in the range of 10 to 20 percent of the body weight.

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Recently a group of organophosphorus compounds has appeared as a new type of very powerful inhibitors of esterase, some of which being extremely toxic especially for nervorus systems. Among them, some are specific inhibitor of enzymes capable of splitting ester linkages, trypsin, chymotrypsin and thrombin.
However, the lethal effect must be due as well to the inhibition of cholinesterase. The present report of our serial research is concerned with the inhibition of some of the insecticides on the activity of carp blood cholinesterase; and its results are summarized as follows:
1) Inhibition as much as 48-54 percent of serum cholinesterase activity was observed at 1-25ppm of parathion which is the lethal concentration for carp; and the same could be said with the one from erythrocyte. Parathion less than 0.001ppm did not show any inhibitory effect on the activity of these cholinesterases (Fig. 1).
2) Malathion at levels 1-25ppm inhibited the 20-30 percent activity of cholinesterase purified from serum. But any inhibitory effect on the activity of the cholinesterase could not be found out at the concentration of malathion less than 0.01ppm (Fig. 2).
3) Inhibition of serum cholinesterase by endrin was so low as 2-8 percent even at its lethal concentration (0.05-0.005ppm) (Fig. 3).
These facts may suggest the possibility that the carp blood cholinesterases are not inhibited by endrin which is one of the organic chlorine insecticides commonly used in our country.

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The hemoglobin (Hb) of eel, Anguilla japonica, was electrophoretically found to consist of a fast component (named F, but whether this component may be separated further into two or more components or not, is not clear at present) and a slow one (named S). Both components were successfully crystallized after isolation either by starch block electrophoresis or by CMC column chromatography (Fig. 1), and were compared to each other in respect of some characteristics. Absorption spectra of component F in met form in visible region, as well as in most derivatives in near-ultraviolet region, were located at longer wavelengths than those of component S or mammalian Hb's (Figs. 2 and 3, Tables 1 and 2). It was also noticed that extinction coefficients of component F at absorption maxima were extremely small in met form. Prosthetic groups of these two components seemed, however, to be the same, protoporphyrin IX.
The solubility in concentrated phosphate buffer (pH 6.6) of component F was far less than that of S, being least among those of Hb's of fishes so far tested⁶⁾ (Fig. 4).
Component F was much more heat-labile both in met and cyanmet form than S (Fig. 5). The heat stability of component F was comparable to that of mackerel Hb, the most labile Hb ever found for fish⁷⁾, while component S was found to be one of the most stable Hb's⁷⁾. Also regarding alkali resistance, component F was more labile than S (Fig. 6). Shape of the denaturation curve differed also between them, the curve of component F showing a sharp break in early phase of the reaction.
Component F was electrophoretically homogeneous below pH 7.5, differing from component S which showed a single component over all the pH range covered (Fig. 7). From the pH vs. mobility curves (Fig. 8), the isoelectric points of components F and S are found to be pH's 5.7 and 7.8, respectively. The latter of such a high isoelectric point seems to be rare among Hb's of vertebrates ¹²⁾.
Column chromatographic behavior on CMC was also studied for both components. As pH of the developer (0.01 M phosphate buffer) was lowered, moving velocities of these components were decreased, at pH 6.5 that of component S being made almost to zero, while F not. Such a difference in chromatographic behavior may be applicable to quantitative separation of these two components.

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Oxygen equilibrium of components F and S, which were separated from each other by starch block electrophoresis, was studied by the spectrophotometric method²⁾. The BOHR effect on the oxygen dissociation curve was clearly different between components F and S (Figs. 3 and 4), the oxygen pressure at half saturation (p₅₀) of the former increasing considerably with decreasing pH, while that of the latter was little affected (Fig. 5). The close relation between the magnitude of the BOHR effect and the number of free sulfhydryl groups of Hb⁴⁾, was ascertained also with these components (Fig. 6). In the case of component F, the sigmoidal character (n value in the HILL's equation > 1) of the dissociation curve at higher pH's changed into the hyperbolic one (n ?? 1) at lower pH's, whereas n for S remained at a constant value of about 2.3 (Figs. 7 and 8). These differences in the mode of the BOHR effect may be responsible for the discontinuity of dissociation curve observed in the case of hemolyzate with pH values below 7 (Fig. 2). The oxygen affinity in the physiological pH region differed between both components, p₅₀ of component F being larger than component S.
Effect of temperature on the dissociation curve was remarkable for both components, p₅₀ of which increased with increasing temperature (Figs. 10 and 11). Heat of oxygenation per mole oxygen for both components was comparable to that of mammalian Hb⁸⁾ (refer to Fig. 12). Effect of phosphate buffer concentration was more remarkable on component F than on S, in the same way as the BOHR effect (Figs. 13 and 14). The n value of each component decreased noticeably with increasing concentration of the buffer, to which may be partly due the crossing of two dissociation curves at concentrations of 0.5 and 1.0M.
These facts were strongly reminiscent of the corresponding components of chum salmon²⁾ Components F and S of the two kinds of fishes may be compared to the Hb of marine fish (lower oxygen affinity and stronger BOHR effect) and that of fresh-water fish (higher oxygen affinity and poorer BOHR effect), respectively.
Blood specimens from some individuals were spectrophotometrically determined of relative amounts of both pigments (Fig. 1). There was a close relation between the proportion and body length, the proportion of component S decreasing with increasing body length, which is in a striking contrast to the case of chum salmon (Table 1). It may be guessed that such a difference stems from the ecological difference between both fishes, i. e., that eel is a catadromous fish, while chum an anadromous one.

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Alkali denaturation curves of hemoglobin (Hb) and myoglobin (Mb) of fishes were determined mainly in oxy form under various conditions. The results obtained were as follows.
Denaturation curves of oxy-Hb of the fishes except flathead appeared between those of horse (fairly resistible Hb among mammalian Hb's^{2, 10)}) and human Hb (least resistible Hb^{2, 10)}) (Fig. 7). Therefore, there seems to be no clear inter-specific specificity of Hb between fishes and mammals in the alkali resistibility. Specificity may be pointed out, however, on the shape of the denaturation curve; the curves of mammalian Hb's were almost straight, indicating that the denaturation was of the first order reaction, whereas those of fish Hb's were not.
The alkali denaturation curves of fish Hb's were different considerably from one another, both in the slope and in the shape of them. Thus the alkali resistibility may be regarded as one of the representations of intra specific specificities of fish Hb.
Though Hb's of many fishes showed gently curved lines, those of some fishes such as carp, mullet, big-eye tuna, and chum salmon presented sharply bended lines, just as suggesting that they are consisting of two components different in alkali resistibility. Hb's of carp and mullet are, however, electrophoretically homogeneous⁶⁾. Although two Hb's were seen also on electrophoresis in the case of chum⁶⁾, the proportion of both components as estimated from its denaturation curve was not agreeable with that calculated from the electrophoretic pattern. In addition, as seen in the case of carp (Fig. 6), the shape of the denaturation curve was markedly different at various pH's. Because of these facts, the alkali denaturation method may be not so useful to analysis of multiple Hb's of fish.
Met-Mb of tuna was much more resistant to alkali denaturation than met-Hb of the same fish (Fig. 10). In order to investigate whether the difference in alkali resistance between both pigments of tuna is applicable to the simultaneous determination of them as previously described in the case of human⁷⁾, experimental conditions (such as derived form of pigments, pH, and time of reaction) under which Mb remains native and Hb denatures completely, were looked for in vain (Figs. 9 and 11, Table 1).

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The autoxidation velocity of myoglobin (Mb) were investigated on crystalline specimens from fishes (two species of tuna, and skipjack), sei whale and horse. The results obtained were as follows.
The autoxidation reaction of Mb, either from fish or from mammal, was of the first order with respect to unoxidized Mb (e. g., Figs. 2 and 4-6). The modes of effects of salt concentration, pH, and temperature on the autoxidation velocity were roughly common to all the species: The velocity constant (κ) was doubled by the increase of phosphate concentration from 0.2 M to 0.6 M (Fig. 3), and so was the case with the decrease of 0.3-0.4 pH unit (Fig. 7). The temperature coefficient was 4-5 (Table 1, Fig. 7). The velocity constant in the autoxidation was not so different among two species of tuna and skipjack, or between whale and horse. And the κ value for Mb of fish as a whole was 2.5-4 times larger than that of mammalian Mb. Mb's of tuna and horse showed maximum κ values at an oxygen pressure of 3.3-4.0mm.Hg (tuna) and 1.7mm.Hg (horse), which were close to the pressures for them at half saturation with oxygen, respectively (Fig. 10). The ratio of the maximum κ value to that at the atmospheric pressure was about 1.7 in the case of tuna Mb, which was far small, compared with about 3.6 in the case of horse Mb. That is, the effect of oxygen pressure on the autoxidation of Mb was slighter for tuna than for horse.

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The characteristics of cephalin (the lipid which is insoluble in acetone and ethanol, and soluble in petroleum-ether) in the ordinary meat and the red meat of “Kan-Buri” (yellowtail, Seriola quinqueradiata, caught in winter) and “Higan-Buri” (yellowtail caught in spring) were compared.
In each sample fish, the yield (%) of cephalin from the red meat was higher than from the ordinary one, and the unsaturation of conjugated fatty acids in the former was higher than in the latter. In both cases of the ordinary and the red meats, the unsaturation of conjugated fatty acids in cephalin was higher in “Kan-Buri” than in “Higan-Buri”.
It is presumed that the main constituents of cephalin in yellowtail are phosphatidyl ethanolamine, phosphatidyl serine and inositol phosphatide, and a small quantity of acetal lipid seems to be contained in cephalin.
The paper chromatography indicated the presence of myristic, palmitic, stearic, arachidic, zoomaric, oleic, eicosenoic, erucic and linoleic acids in cephalin. The main constituents of the saturated fatty acids in cephalin were palmitic and stearic acids, and the main constituents of the unsaturated acids in it were oleic and zoomaric acids.

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The defatted test of Cynthia roretzi v. DRASCHE contained 42.5 per cent of crude protein (Table 1). When the test was autoclaved with water at 20 pounds for 2 hours, about one-fourth of its total nitrogen dissolved in water (Table 2).
In the enzymic digestion of test, about 40 per cent of the total nitrogen was hydrolyzed by pepsin or papain but only 1.1 per cent by trypsin. On the contrary the denatured test formed by autoclaving was remarkably digested at the level of 67.7 per cent by the last enzyme (Table 3).
The amino acid composition of the scleroprotein was similar to that of ichthulokeratin and neurokeratin (Table 4). It showed low contents of cystine and serine as compared with eukeratin, and differed sharply from elastin and collagen rich in glycine, alanine and valine and poor in cystine and tryptophan. The ratio of histidine: lysine: arginine in this scleroprotein was 1:2.9:4.2, which agrees with that of ichthulokeratin and differs from 1:4:12 of eukeratin.
These results led the authors to classify this protein as a pseudokeratin.

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A water soluble protein was isolated from the test of Cynthia roretzi v. DRASCHE by extraction with water and subsequent purification by repeated precipitation with acetic acid and ethanol. It was found to give a single spot or zone on paper electrophoresis, (Fig. 1), and was positive to color reactions of protein and carbohydrate, (Table 2), and was precipitated by various protein precipitants, (Table 3). It contained 10.39 per cent of nitrogen and 16.28 per cent of hexosamine (as glucosamine). The amino acids and sugars of the hydrolysates were qualitatively detected by paper chromatographies as follows: cystine, lysine, histidine, arginine, glutamic acid, aspartic acid, glycine, alanine, serine, threonine, valine, leucine, phenylalanine, tyrosine, proline, methionine and tryptophan, and mannose, galactose, glucose, xylose, rhamnose and fructose.
Therefore, it was proved that the water soluble protein of the test is a mucoprotein.

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The authors determined the general chemical components of the ascidian, Cynthia roretzi V. DRASCHE collected at the Onagawa Bay in Miyagi Prefecture. The results obtained were as follows:
1. The height, width, total weight, test weight, muscle weight and viscera weight of the ascidian were 4.5-13.4cm, 2.7-8.lcm, 17-195g, 11-139g, 3-50g and 2-21g, respectively, (Table 2).
2. The contents of moisture, crude protein, crude fat, crude ash and glycogen were 75.12 ± 1.61%, 9.36 ± 0.85%, 2.35 ± 0.41%, 1.65 ± 0.21% and 11.56 ± 1.42% in the muscle, and 78.31 ± 1.64%, 13.19 ± 0.87%, 3.19 ± 0.44%, 1.75 ± 0.41% and 3.52 ± 0.93% in the viscera, respectively, (Table 3). It should be remarked that the ascidian contained a large amount of glycogen compared with other marine invertebrates such as mollusca and crustacea.

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In industrial practice of molecular distillation of vitamin oil it is generally very hard to expect the operation to be carried out with constant efficiency since the vitamin A as the object matter is often subject to great fluctuation in regard to magnification of concentration, yield and etc. As reported in this paper, the author conducted researches hence into some factors to be regarded as main causes of the above-mentioned fluctuation and tried to obtain fundamental knowledges necessary for carrying out industrial molecular distillation of vitamin oil with constant efficiency.
In the first place, method of pre-refining the vitamin oil to be subjected to distillation being examined, the results shown in Tables 1, 2 and 3 were obtained. These results indicate that the effect of pre-refining reflected in the results in subsequent distillation varies not only with the kind of pre-refining method but also with that of raw vitamin oil subjected to pre-refining as follows: In the case of liver oil of pollack, three methods here examined stand in respect of refining effect in the order of alkali-refining > washing with water > treatment with diatomaceous earth. In pre-refining liver oil of dogfish, three methods applied thereto, namely, alkali-refining, washing with water and treatment with active charcoal, are not greatly different from one another, leading equally to rather good results. In the case of whale liver oil, with which the effect of combination of alkali-refining with subsequent washing with water was examined, there can be got ready for the better result in molecular distillation by the more frequent washing with water, an especially good result being obtained when the oil is treated first with alkali and then with aqueous citric acid.
As to the color of the oil fraction remaining after the end of molecular distillation, it may be expected from the data in Tables 1 and 2 that a great improvement can be realized by pre-refining the raw oil with active charcoal.
In the second place, the phenomenon of sludge formation which causes the distiller to become undesirably stained was observed by carrying out molecular distillation experiments in a semi-micro pot still, the results shown in Table 4 and Fig. 3 being obtained. As seen from these results, sludge formation of vitamin oil begins at a temperature of 260-280°C and becomes remarkable at 300°C. Among the samples here examined, tuna liver oil was most liable to produce a serious amount of sludge and pollack liver oil came next, while liver oil of dogfish went into sludge only in a small proportion.
In the third place, the so-called out-gas, namely, gaseous decomposition products given rise to as vitamin oil is subjected to molecular distillation at an elevated temperature was examined of its amount of evolution by the help of the displacement curve 2 shown in Fig. 2. As seen from the results given in Table 5, the amount of out-gas evolved in the working stage of the 1st degassing was very large irrespective of the kind of animal from which the sample oil was obtained, every 1kg oil specimen giving off the out-gas amounting to 1, 500lmmHg under a vacuum of 2mmHg and 2, 400lmmHg under that of 3mmHg. Amount of out-gas was appreciably reduced, however, in the working stage corresponding to the 2nd degassing, the amount dropping to about 170lmmHg and about 80lmmHg with whale liver oil and with fish liver oils, respectively. Evolution of out-gas can prevail also in the working stage of distillation proper as seen from the result that the amounts of out-gas evolved in one run of feeding the distiller with vitamin oil were not less than 337-265lmmHg, 280-235lmmHg and 235-155lmmHg in the cases of liver oils of whale, “yoshikirizame” (Prionace glauca) and cod or dogfish, respectively.

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