February, 1926
INDUSTRIAL A N D ENGINEERING CHEMISTRY
rent is broken and the sample allowed to cool for 3 minutes. After cooling, the contraction in millimeters, indicated on the scale, is read. PRECAUTIONS-The walls of the buret and combustion chamber should be kept slightly moistened t o insure accurate results. Water should be kept above the point at which the platinum wires are sealed into the glass stopper, since any leaks around the stopper or seals can be detected readily.
Calibration of Apparatus
The results obtained by use of the apparatus are empirical and their interpretation involves the use of a calibration table or chart. The fundamental factors involved in working u p such a table or chart are ably and fully discussed
185
by Jones and The chart employed in the present work is similar to those described by Jones and Yant. The method of calibrating the N-D apparatus has been to prepare mixtures of air with varying proportions of hydrocarbon vapor, and to determine the composition of these mixtures with the Haldane set, figuring the percentages of combustible material on the basis of the chemical equation characteristic of pentane. Portions of the same mixtures were tested in the N-D apparatus and the contractions (in millimeters) determined, which were equivalent to the various percentages of hydrocarbon vapor, calculated as pentane. These figures are used in preparing a chart such as is shown in Figure 2. This chart is used to convert the contraction readings into percentages of combustible vapor.
Another Important Role Played b y Enzymes in Bating’ By John Arthur Wilson and Henry B. Merrill A. F. GALLUN& SONS Co.,MILWAUKEE. Wis.
During the liming of skin for tanning, keratin is thus rendering the N M O D E R N tannery partially broken down into keratose, which is soluble grain surface of the finished practice, after the skins in neutral or alkaline solution, but is precipitated at l e a t h e r finer, softer, and have been l i m e d , u n its isoelectric point, pH = 4.1. If this material is c l e a r e r in color, although haired, scudded, and washed, left in the skin and precipitated there by the acid tan Rohm and Haas’ have sugbut before they are tanned, liquors, the value of the finished leather is lowered. gested that probably a more they are subjected to a procIn bating, keratose is broken down by the enzymes important function consists ess called bating, which conto a point where it is no longer precipitable by acid. in dissolving out any remains sists in paddling tJhe skins, The rate of digestion of keratose is directly proportional of u n o r g a n i z e d material still plumped by the alkaline to the concentration of enzyme and is a maximum at known as scud, which is delime liauor, in a warm soluPH = 7.9. r i v e d f r o m t h e epithelial tion ofpancreatin and buffer tissues and is insoluble in salts designed to keep the pH value a t about 8 until they have been reduced from a neutral or alkaline solution. Wilson and Daub demonstrated conclusively by means of plump to a flaccid condition.2 Bating is an extremely complex process and one which is difficult to operate efficiently photomicrographs that the elastin fibers of calfskin can be at all times. Limed skin, with its manifold and variable pro- completely removed by solutions of pancreatin without tein degradation products, may be acted upon by enzyme appreciable damage to the collagen fibers. I n fact, the only bate liquors, with their countless bacteria, in many different action of the enzymes in bating which they could detect ways. Depending upon the kind of skin, its previous treat- under the microscope was the removal of the elastin fibers. ment, and the kind of leather desired, bating may serve one However, the writers’ experience since that time has demonor more very useful purposes, or it may be without appreci- strated clearly that the beneficial effects of the enzymes in able effect, or i t may actually prove harmful. Moreover, bating are not always measured by their power to digest where bating is desirable, the method of operation must elastin. If the dissolved keratose which is present in limed be varied to correspond with any variations in previous skin is not removed before tanning, it will be precipitated treatment of the skin. in the skin by the acid tan liquors, with consequent damage It has been the aim of the writers to ascert:tin all of the to the appearance of the grain surface of the leather. The useful purposes which bating may serve, to study the mechan- soluble keratose is much more quickly broken down by the ism of the reactions involved, and to devise means for con- enzymes of the bate liquor than the insoluble elastin fibers, trolling each reaction so that the particular purpose may be and it was found in numerous tests that nearly all measurable served most efficiently. Already five different purposes effects of the bating of certain types of calfskin were obtained of bating have been recognized and means for their quanti- before the elastin fibers had been measurably attacked. tative control indicated: (1) rendering the skin flaccid; Thus the removal of keratose must be added to the list of ( 2 ) adjusting the hydrogen-ion concentration of‘ the solution important functions of bating, and the following report deals adhering to the skin; (3) removal of lime; (4) removal of with the quantitative relations involved and with the measelastin fibers; and ( 5 ) partial digestion of collagen. To urement of the keratose digesting power of bating materials. these the writers are now adding a sixth, the removal of Preparation of Keratose keratose, and this forms the subject matter of the present The material in which we are interested consists of those paper. decomposition products of keratin which are soluble in alThe prevailing opinion has been that the chief function Rosenthal, J . A m . Leather Chem. Assoc., 11, 463 (191ti). of the enzymes in bating is to digest the elastin fibers of the
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Krall, Socidt~Anonyme anc. B. Siegfried, private bulletin, 1918. Seymour-Jones, J . Sac. Leather Trades’ Chem., 4, 60 (1920). e Wilson and Daub, THISJOURNAL, 13, 1137 (1921). 7 J . A m . Leather Chem. Assoc., 18, 516 (1923). 4
Received October 15, 1925. 2 For a detailed account of this process see Wilson, “The Chemistry of Leather Manufacture,” p. 173.
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kaline solution and precipitated upon acidification. The term “keratose” has been limited to this material in consequence of which the keratose content of the prepared solutions may be defined as the weight of material precipitated by the addition of acid to the point of maximum precipitation. The keratose used in these experiments was prepared by digesting thoroughly cleansed calf hair with ten times its weight of 2 N sodium hydroxide for 18 hours a t 25’ C.
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Figure 1-Isoelectric Point of Keratose Indicated by Point of Maximum Precipitation
The resulting solution was treated with hydrochloric acid to reduce the pH value to 8, at which the keratose is still soluble. The solution was filtered and then acidified to p H = 4, a t which point the keratose was precipitated as a voluminous, white mass, such as is seen when titrating a used lime liquor with acid. The precipitate was allowed to settle in tall cylinders and washed repeatedly by decantation. Finally, it was redissolved by adding sodium hydroxide to make the p H value of the solution equal 8. This solution was preserved with a small amount of thymol and kept in the refrigerator until used.
Vol. 18, KO. 2
At pH values still further removed from the range no precipitate was formed a t all. It is rather important to note that the isoelectric point of keratose corresponds closely to the pH value of many tanning solutions. Experimental Procedure
The immediate object of this investigation was to determine the rate of hydrolysis of keratose by pancreatin as a function of pH value, time, and concentration of both enzyme and keratose. The keratose and enzyme solutions were mixed with Sorthrop’s citrate-phosphate-borate buffer solution to which hydrochloric acid or sodium hydroxide had been added to produce the desired pH value in the digesting mixture. The object of the buffer solution was to maintain constancy of p H value during the digestion, and electrometric measurements made before and after each digestion showed that this was accomplished. It was also demonstrated that the presence of the buffer material did not introduce any complications, since series run without added buffer gave results more erratic, but otherwise similar in value to those obtained with buffer material. The digestions were made a t 40” C. At the end of the digestion period the undigested keratose was precipitated by the addition of enough acetic acidsodium acetate mixture to bring the p H value to 4.1, filtered off, dried, and weighed on a tared filter paper. The pancreatin used was from the same parcel as that used and described by Wilson and Daub.6 Effect of pH Value
Figure 2 shows how the hydrolysis of keratose varies with pH value in the presence and absence of pancreatin. Subtracting the values in curve B from those in curve A , we get curve C, which represents the extra hydrolysis due to the added enzyme. At pH values below 5.5 and above
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gram per l i t e r : k e r a t o s e - --1.670 panc r e a t i n- o .om temp. 40°C.
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Isoelectric Point
The isoelectric point of keratose was measured by the pH value causing maximum precipitation. Keratose solution, containing approximately 2 per cent by weight of keratose, was mixed with an equal volume of the powerful citrate-phosphate-borate buffer solution described by Sorthrope and 8 volumes of water. One hundred cubic centimeter aliquots were pipetted into 50-cc. portions of acetic acid solutions of different concentrations to give pH values covering the range of maximum precipitation. The precipitate formed in each case was filtered off, dried, and weighed. The maximum amount precipitated in any test was taken as the total keratose content and the percentage precipitation in the other tests was calculated from this. Two entirely independent series of tests were run and both are plotted in Figure 1. The point of maximum precipitation is fairly sharply defined a t pH = 4.1. It is believed that this is the first time a figure has been reported for the isoelectric point of keratose. An attempt was made to cover a greater range than that shown in Figure 1, but a t p H values just outside this range the precipitate was so slimy as to be practically unfilterable. 8
J . Gen. Pkysiol., 5, 263 (1923).
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of pH Value on Digestion of Keratose by Pancreatin
11.2, the enzyme is without effect, while it shows its optimum action a t pH = 7.9. The range of comparatively great activity extends from about 7 . 5 to 8.3, which is the range ordinarily found in bate liquors. Effect of Time
The progress of the digestion of keratose by pancreatin with time is illustrated in Figure 3. For comparison, a blank and a solution of a typical commercial bate are included. The curves are typical of protein hydrolysis by
ILDUSTRIAL A S D E,VGI.VEERISG CHEMISTRY
February, 1926
187
1.535 gram k e r a t o s e p e r l i t e r .
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temp, 404C. pH = 7.9. 1.64 grams k e r a t o s e p e r l i t e r ,
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Grams Pancreatin per L i t e r Figure 4-Rate
of Keratose by Enzymesas a Function of Time
enzymes and the curve for the blank shows that during the first 24 hours practically all the digestion is due to the enzyme. Effect of Concentration of Enzyme
With enzyme content the only variable, it was found that the time required to hydrolyze a given fraction of the keratose is inversely proportional to the concentration of enzyme. This is shown strikingly by the straight lines in Figure 4, where the reciprocal of the number of hours required to digest 20, 30, and 40 per cent of the keratose, respectively, is plotted against the initial concentration of the enzyme.
of Digestion of Keratose a8 a Function of Concentration of Pancreatin
move elastin, another to digest some of the collagen, and another the keratose. If given a dozen different enzyme samples to select from, it is probable that each would find a different one to be the most active bating material for his
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temp. 40°C. pH = 7.9.
Effect of Concentration of Keratose
For a given initial concentration of enzyme, the rate of digestion increases with the value for the initial concentration of keratose. This is indicated in Figure 5, where the time required to hydrolyze a given fraction of the keratose present is plotted against the initial concentration of keratose. Where 2 grams per liter of keratose are present 0.8 gram is hydrolyzed in 135 minutes, but where 4 grams per liter are present 0.8 gram is hydrolyzed in only 93 minutes.
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Comparison of Proteolytic Powers of Different Enzyme Preparations
Attempts have been made to determine the bating power of enzyme preparations by measuring their power to hydrolyze gelatin or casein, but it has been found that one cannot judge the proteolytic power of an enzyme preparation as regards one kind of protein from its action upon another protein. For example, on experimenting with two enzyme preparations both of which hydrolyze keratose, it is found that one of them is apparently without effect upon elastin, while the other hydrolyzes it readily. Commercial enzyme preparations are usually mixtures of several enzymes, which may be present in very different proportions in different preparations. Thus the sample showing the higher proteolytic power for one protein may show a lower power for another protein. One tanner may bate his stock to re-
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Figure 5-Time Required t o Digest a Given Fraction of Keratose as a Function of Initial Concentration of Keratose
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purpose. The proteolytic power of each sample must be tested upon the particular substrate for which it is t o be used, or upon more than one where it is to be used to digest several kinds of proteins. The writers, method for measuring the keratose digesting power of an enzyme preparation is as follows: From a stock solution of keratose, take an amount containing 2 grams of dry keratose, add the citrate-phosphate-borate mixture and acid or alkali to make a final pH value of 7.9, heat to 40' C., and then add the enzyme dissolved in enough water a t 40' C. to make a total volume of 1 liter. Keep the mixture a t 40" C. and withdraw 100-cc. portions a t intervals, mix with acetic acid-sodium acetate solution to give a final pH value of 4.1, which precipitates the undigested remains of the keratose, and filter. Then dry and weigh the precipitate. Withdraw the first portion immediately after the mixing to obtain the amount of keratose present a t zero time. Withdraw the remaining portions after 0.5, 1, 2, 4 , and 8 hours, or after a longer period i f necessary. From the results plot a curve showing the fraction of keratose digested as a function of the time. From this curve pick the time corresponding t o the digestion of 40 per cent of the keratose initially present. Then take the keratose digesting power as the value l/hg, where h is the time in hours required to digest 40 per cent of the keratose and g the number of grams of the enzyme preparation per liter. The constancy of
Vol. 18, No. 2
this ratio for a given sample is illustrated in Figure 4. In each test take an amount of enzyme such that the point of $0 per cent digestion occurs between the first and eighth hours.
For the pancreatin used in this work, a solution containing 0.1 gram per liter will hydrolyze 40 per cent of the 2 grams per liter of keratose in 2.2 hours a t 40" C. and at pH = 7.9. Its keratose digesting power is therefore 1/(2.2 X 0.1) = 4.54. The values for other enzyme preparations submitted
to the writers as bating materials range from 0.06 to 86.0. Summary
An important function of bating is the removal of keratose from the skins by hydrolysis with enzymes. The isoelectric point of keratoseis a t p H = 4.1. . Hydrolysis of keratose by pancreatin proceeds only in the pH range 5.5 t o 11.2 and is a maximum a t pH = 7.9. The hydrolysis a t 40" C. and pH = 7.9 has been measured as a function of time and concentration of enzyme and of keratose. A satisfactory method has been outlined for the determination of the keratose digesting power of bating materials.
Vitamin Potency of Cod-Liver Oils' XVIII-Effect on Vitamin Potency of Cold-Pressing Cod-Liver Oils By Arthur D. Holmes and Madeleine G. Pigott THEE. L. P A T C B Co.. BOSTON,
IX typical crude cod-liver oils of medicinal grade were
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pressed under commercial conditions by refrigeration in a brine-cooled tank. The solidified stearin was separated from the nonfreezing oil by means of a Bay press equipped with medium-fine canvas filters. Representative samples were taken of the crude and pressed oil. These were analyzed by the usual chemical method to determine the nature of the oils under consideration. Comparing the analyses, it appears that there is little difference in specific gravity of the two oils, that the refractive index and iodine values are slightly higher for the pressed oil thaq for the crude oil from which it was obtained, 1
Received September 17, 1925.
MASS.
and that the saponification and acid values vary for different oils. Considering the analyses as a whole, there appears to be little difference between the chemical and physical characteristics of crude and pressed cod-liver oils of the type under consideration. The vitamin potency of the oil under consideration wad determined by the usual laboratory procedure which has already been described in detaiL2 Six experimental animals whose body reserve of vitamin A had been exhausted were assigned to each of the oils to,be tested. The oil was fed separate from the experimental diet in amounts varying from 2
J . Metabolic Research, 2, No. 3 (September, 1922); THIS JOURNAL,
16, 1181 (1924): 17, 75 (1926).
