March, 1923
INDUSTRIAL A N D ENGINEERING CHEMISTRY
267
Pancreatin as an Unhairing Agent' By John Arthur Wilson and Albert F. Gallun, Jr. LABORATORIES OR A. F.GALLUN & SONSCo., M I ~ W A Y K EWIS. E,
N 1913 Rohm2described
I
When calfskin is swollen in dilute NaOH, neutralized with PRELIMINARY PROCEDURE a process for unhairNaHCO,, and then put into a suitable solution of pancreatin at 25 C. ing and bat,ing skins in The studies were made exposed to air, the hair is completely loosened, but the action is apone operation, involving upon pieces of fresh calfparently not due to the enzyme, since it is checked by cooering the the use of an alkaline soluskin, about 5 x 3 in., which solutions with a layer of toluene. Neither does the enzyme produce tion of pancreatin, Since had been thoroughly soaked a bating action at 25" C. then pancreatin has often and cleansed. Each exAt 40' C . a solution of pancreatin fails to cause a loosening ofthe been listed as an unhairing periment was carried out hair of fresh skin. apparently because the corneous layer of the agent, although in the disboth a t 40" and a t 25" C. epidermis is impermeable to the enzyme and thus prevents it from cussion following the preThe action of the enzyme reaching the soft Malpighian layer below. sentation of Rohm's paper, solution upon the skin in When a skin has preoiously been swollen by acid or alkali, and E b e r 1 e expressed the each test was compared the corneous layer thus rendered softer and more permeable, a suitaopinion that an aqueous with the action of a blank ble solution of pancreatin at 40 C., eoen under toluene, will not on19 identical with the enzyme extract of the pancreas does destroy the Malpighian layer of the epidermis and loosen the hair, not loosen the hair, that solution, except for the fact but it will also dissoloe the elastin fibers of the skin, effecting both that it contained no enin Rohm's preparation no unhairing and bating in a single bath. zyme. This solution was pure pancreatin was used, and that the action was prepared by diluting 18 cc. probably due to the accompanying material. As far as we of N NaOH and 2.8 g. of NaH2P04 to 1 liter, and all enzyme are aware, little effort has been made to determiqe the spe- solutions were made by adding to it 1 g. of pancreatin per liter. The p H values did not vary more than 0.1 from the cific role played by the enzyme. This process has been used to some extent on a practical value 7.6 in any case. The enzyme solutions and blanks, scale, and in 1920 Hollander3 described it as having many as well as solutions used for the pretreatment of the skin, advantages over the old system of liming, and claimed that were all covered with a layer of toluene to check bacterial it depends entirely upon enzyme action for unhairing. Ac- action. The results were checked with pieces of skin from cording to his description, the skins are first soaked for one different calves. day in dilute sodium hydroxide solution and then transferred ACTIONOF PANCREATIN ALONE to a dilute solution of sodium bicarbonate, to which the enzyme is added after the swelling due to the alkali has been The effect of pancreatin upon skin not previously soaked counteracted. Twenty-four hours later the hair is com- in sodium hydroxide solution or any other swelling agent pletely loosened and can be rubbed off. was studied first. After 24 hrs. of contact of skin and soluA preliminary examination of the method was made in tion, little action was noticeable either a t 25" or 40" C., but this laboratory by soaking pieces of thoroughly cleapsed calf- after 48 hrs. the collagen fibers of the skin in the enzyme soskin in 0.05 N hTaOHfor 1 day, replacing the solution the next lution a t 40" C. began to dissolve very rapidly, the action day by 0.1 N NaHC03, and 5 hrs. later transferring the pieces proceeding from the flesh side, but there was no indication to a solution made by diluting 18 cc. of N NaOH, 2.8 g. of of the hair loosening. On the other hand, the skin in the NaH2P04,and 1 g. of U. S. P. p a n ~ r e a t i nto , ~ 1liter. The p H blank a t 40" C. and those a t 25" C. in both blank and enzyme value of the solution was found to be 7.52 at 25" C., lying well solution still remained but little affected. It was evident within the range of optimum activity of this enzyme. Two that pancreatin has a more powerful solvent action upon experiments were run at a temperature of 25" C., but in one collagen fibers than upon the epidermis of a skin not prethe solutions were left exposed to air, as would be the case in viously swollen with acid or alkali. The time factor involved practice, while in the other they were covered with a layer of in the destruction of the collagen fibers is interesting. The toluene to check bacterial action. After the pieces had been action seemed to indicate that the fibers are coated with in the enzyme solutions for 24 hrs., the hair of the pieces some material more resistant to tryptic digestion than the from the solutions exposed to air could be rubbed off with collagen beneath it. The term LLcollagen" is used in this paper great ease, leaving the grain membrane clean and white, but in its general sense to include all the skin protein convertible that of the pieces from the solutions under toluene remained into gelatin. Possibly this covering may be found to bear firmly fixed. This seemed to indicate that the unhairing some relation to the fiber "sarcolemma" discovered by Alfred action obtained a t 25" C. was not due to the enzyme, but Seymour-Jones.5 probably to proteolytic bacteria or their products. Wilson and Daub4 found pancreatin to have no visible The doubt thus cast upon the role played by pancreatin in solvent action upon the collagen fibers of calfskin during the this method of unhairing made it desirable to carry the in- bating process. Thomas and SeymourJones,6 on the other vestigation further, and particularly to examine the action of hand, found that hide powder is digested by pancreatin. pancreatin a t 40" C., the temperature of its maximum This erfperiment reconciles these findings, which a t first apactivity. peared contradictory. 1 Received August 2, 1922. Presented before the Division of Leather Chemistry at the 04th Meeting of the American Chemical Society, Pittsburgh, Pa., September 4 to 8, 1922. 2 "A New System of Liming,'' Collegzum, 1918, p. 374; J . A m . Leather Chem. Assoc., 8 (1913), 408. 0 "Unhairing Hides and Skins by Enzyme Action," J . A m . Leather Chem. Assoc., 15 (1920), 477. 4 Sample described by Wilson and Daub, THIS JOURNAL, 18 (1921), 1137.
PRETREATMENT WITH SODIUM HYDROXIDE In the next series of experiments, the pieces of skin were kept for 24 hrs. in 0.05 N NaOH at 25" and 40" C . ,respectively. 6 6
"Physiology of the Skin," 1.SOC.Leather Trades' Chem., $3 (1918). 203. Advance note.
*
268
INDUSTRIAL A N D ENGINEERIh'C CHEMISTRY
Val. 15, N o 3
Fie. Z - ~ ~ T I C A L S ~ C T I O SXOW~NO N R S O ~ D OF N HMR Foirrcris os Fcc: I-VSRIICAL S B m i o ~Saowrmo REGION 0s IIma FO,LLICLE$S 0m IN 0.05 N NaOIl lion 1 DAYANII THEN FOB 1 DAYIN 0.1 C ~ , s s ~ SOAKBD m m 0.05 N NaOH POR 1 Dnu AND THBNPOX 1 DAYIN C a ~ s s ~SOAXBD PBn E B N ~PBNC~EATIN SOI-VT~OW AT 40' C., U N D R R Toiusnn. Txe 0.1 YBn CENT P A N C B 4 A T m S O L U T ~ NIT 2 5 O C., E n e o a ~ oTo Ai.. Tnn F r a s ~ sIIAYEDISAPPBAIIID ALONG ~ i r i rTBS E ~ m e n ~ i sMAG. EP~DBBM A N~ D SS B ~ A C S OGLANDS UE H*vi DISINTEORATED. a m TISB 3 ; ~ ~ s - BLIET~N NIBIFATION 145 D I A M E T B I S n~ Fissna Ans STILL Irrracr A N D APPZAX6s D m n T x a s ~ n RUNNINO s N Z A R S Y HORIZONTALLY. M A E N I ~ I ~ ~145 T ~DIAMETBRZ N
The solutions were then replaced by 0.1 N NaHCOs of corresponding temperatures, and 5 hrs. later by the enzyme and hlanksolut.ions, in which theskins remained for24 hrs. The unhairing action in the enzyme solution at 40" C. was completely satisfactory, an indication that at this temperature pancreatin may be considered an unhairing agent for calfskin previously swollen in dilute NaOH solution. A very slight unhairing action vas noticeable in the blank at 40" C.., evidently due to the previous t.reatment with alkali. No unhairing action could be detected in the bla.nk or enzyme solution at 25' C. PRETREATMENT WITH HYDROCHLORIC ACID The preceding series of experiments was repeated exactly except that 0.05 N HCI was substituted for the alkali as the swelling agent. 4 t 25' C. there was no visible unhairing action either in the blank or enzyme solution. In the €IC1 bath at 40" C. the pieces of skin began to jelly; there was no further change in the piece transferred to the blank R t 40" C., but the piece put into the enzyme solution at 40" C. was quickly destroyed, the collagen passing into solution, leaving the epidermis and hair floating in the liquoy. Tbe opposite effects of acid and alkali upon the skin at 40" C. are interesting. 0.05 N SsOH hydrolyzes the epidermis more rapidly than the collagen fibers, whereas 0.05 N HCl hydrolyzes collagen much more rapidly than the epidermis. The experiment m s repeated except for the fact that the pretreatment vitb RCI m i c done at 25' C. and the digestion vrith pancreatin a t 40° C. Aftcrtl~eshinhadbeen in tbe pan-
creatin solution for 24 hrs., the unliairing action was complete, shon-ing that the effectiveness of pancreatin as an nnhairing agent depends upon the previous swelling of the skin, but regardless of whether the swelling is caused by acid or alkali. The fact that pretreatment with NaOII was done at 40' C. did not seriowly influence the result, for, when another piece of skin was soaked in 0.05 N NaOH a t 25" C. and then in the pancreatin solution at 40' C., the unhairing action was entirely satisfactory. h E l ' R E A T M E K T WITH A\%>tuKIil
Being a mry uxak base, ammonium hydroxide produces much less swelling of skin than NaOH in dilute solution. Ammonia is, nevertheless, a poiverfnl unhairing agent in sufficiently concentrated solution. We h a x been able to nnhair fresh calfskin in a few hours by soaking in 2 N KH40H, The action is not reliable, however, for the ordinary run of skins in commerce, hecause it is influcnccd by t,he previous treatment of the skin; on some skins the ammonia loosened the hair only in pat.ches. Experiments dealing with the action of pancreatin upon skins previously treated with ammonia were carried out exactly like tbose of the SaOH series, except for the replacement of the 0.05 N NaOH hy 0.5 N NH,OR. The hair m e loosened to some extent by t.lie pretreatment with ammonia, more at 40" C. than at 25" C. After the pieces had been in the blank and enzyme solntions for 24 hrs. they all sbomd some unhairing action, hut in no casc n.as it entirely satisfactory. The degree of action might he given a very rough
March, 1923
INDUSTRIAL A N D ENGINEERING CHEMISTRY
rating by calling that in the enzyme solution a t 40" C., 75 per cent, that in the blank a t 40" C., 50 per cent, and that in both blank and enzyme solutions a t 25" C., 25 per cent. Evidently, the pretreatment of skin with ammonia, which is itself an unhairing agent, does not assist the unhairing action of pancreatin nearly so much as pretreatment with materials that merely swell the skin.
COMBINED BATING AND UNHAIRING ACTION The work of Wilson and Daub indicates that the fundamental action of bating is the removal of elastin fibers from the skin. We therefore measured the extent to which the pieces of skin were bated in the pancreatin solutions by observation under the microscope. The two sections shown were cut a t 30p, stained with logwood and bismark brown to make the elastin fibers stand out prominently wherever present, and photographed through a Wratten filter, H (blue-green), using an 8-mm. objective and a 5x eyepiece. When the pancreatin method of unhairing is used commercially the liquors are left exposed to air. Our experiments show that the hair loosening can then be effected a t a temperature of 25" C., but that the action is apparently not due to the enyzme, since it is checked by covering the solutions with toluene. But if pancreatin is not the active agent, we should expect the action not to be accompanied by elastin
269
removal. Fig. 1 corroborates this view; where the hair loosening has been effectedby a pancreatin solution a t 25" C., exposed to air, the epidermis has been disintegrated and the hair separated from the skin, but the elastin fibers remain undissolved. At 40" C. a pancreatin solution, such as those used in these experiments, was found to dissolve the elastin fibers completely from unhaired calfskin in about 7 hrs. I n the unhairing experiments, where the pieces from the enzyme solutions a t 40" C. had not previously been swollen in acid or alkali, microscopic examination showed that all the elastin had been dissolved away from the flesh side of the skin, but none from the elastin layer near the epidermis. The hard corneous layer of the epidermis had apparent,ly acted as a membrane impermeable to the enzyme. I n the ordinary methods of unhairing, such as liming, the unhairing agent acts upon the cells of the Malpighian layer which lie between the corneous layer and the derma. The impermeability of the corneous layer to the enzyme explains why the pancreatin did not attack the Malpighian layer and loosen the hair. I n acid or alkaline solutions, the corneous layer swells considerably and is thereby rendered more permeable. That it is also attacked by the enzyme when in the swollen condition is indicated by the fact that no corneous layer could be found in some of the sections examined.
~~~~
Total and Available Calcium Hydroxide in Hydrated Lime' By A. S. Behrman and F. R. Porter INTERNATIONAL F I L T E R
T HAS BEEN customary to evaluate hydrated lime for water softening by its total content of calcium hydroxide. Experience in the use of hydrated lime in water softening has shown that the quantity of calcium hydroxide actually available and taking part in the water-softening reactions is frequently not the same as the total quantity of calcium hydroxide determined by the usual methods. This discrepancy is due to a number of factors, chief among which are the limited period of enforced intimate contact of particles of lime with the water being softened; the coating of the lime particles, either with a shell of the carbonate resulting from air slaking, or, during the first few minutes of the softening process, with the insoluble products of the reaction; the rate of settling of the lime particles; and the physical nature of the lime particles themselves. The purpose of this note, and the paper of which it is a condensation, is to point out that hydrated lime to be used for water softening should be purchased on the basis of available, not total, calcium hydroxide content; and the plea is made for a method of analysis which will give a result more comparable with the actual value of the hydrated lime for water softening than the usual procedures employed. The following method of analysis is suggested as approximating conditions encountered in water softening. The data so far obtained are very satisfactory; but time has not been available to accumulate sufficient evidence to prove the general applicability of the method. It is published a t this t h e , therefore, merely as a suggestion, since the authors will be unable for some time to do any further experimental work on this method, or on any desirable modifications thereof which may be developed. I n the method suggested the calcium hydroxide converts
I
1 Condensation of paper read before the Division of Water, Sewage, a n d Sanitation a t the 64th Meeting of the American Chemical Society, Pittsburgh, P a , September 4 to 8 , 1922.
CO , C H I C A G O ,
ILL.
sodium bicarbonate to sodium carbonate. One gram of the unscreened hydrated lime is mixed with about 100 cc. of boiled distilled water, and allowed to stand several hours to insure the precipitation of magnesium as magnesium hydroxide. If time does not permit, however, the precipitation may be hastened by heating the mixture for 30 min. a t or near the boiling temperature. To the cooled mixture are now added 400 cc. of approximately 0.1 N sodium bicarbonate, made just neutral to phenolphthalein. The whole is then shaken or stirred for 15 min. and allowed to stand for 33/4 hrs. The mixture is then filtered, and 50 cc. of the filtrate are titrated with 0.1 N hydrochloric acid, using phenolphthalein as an indicator. The number of cubic centimeters of acid times 7.4 equals the per cent of calcium hydroxide. There are indications that the alkalinity due to residual calcium carbonate involves an error of less than 0.5 per cent and so may be neglected. It is probable also that the preliminary period of contact of the lime with pure water may be omitted, and the lime may be added directly to the sodium bicarbonate solution. We have also contemplated a shaking or stirring period of 1 hr. followed immediately by filtration and titration of the filtrate. This method simulates actual conditions encountered in water softening to the extent that the digestion is made with an alkaline solvent, and that there is formed an insoluble product of reaction. The suggested method yields a filtrate containing an excess of sodium bicarbonate. This is not as truly typical as if a slight excess of calcium hydroxide were present. The advantages of the excess of bicarbonate are that the indefiniteness of the calcium hydroxide-sodium carbonate equilibrium is avoided, the solution may be handled without extreme precautions to avoid absorption of carbon dioxide, and the calculation of the results is extremely simple.
