February, 1924
INDUSTRIAL A N D ENGINEERING CHEMISTRY
It is hoped that the current and voltage data given above will be of considerable assistance to others in building furnaces of somewhat modified design. Ease of repacking, heaby electrode connections, positive pressure on the electrodes, and solid construction in general are emphasized.
157
ACKNOWLEDGMENT The author acknowledges the aid of experience gained in the research laboratory of the National Malleable Castings Company, and the assistance of C. F. Block.
Action of Trypsin upon Diverse Leathersls2 By Arthur W.Thomas and Frank L. Seymour-Jones COLUMBIA UNIVBRSZTY, N E W YORK,
N. Y.
tested on a casein substrate, 7’ HAS been shown that It is shown that trypsin is capable of hydroryzing collagen which according to the condition the proteolytic enzyme, has been treated with aarious agents, such as gallotannin, quinone, laid down by Sherman and trypsin, catalyzes the formaldehyde, and copper sulfate. Where the tanning agent comNeun2-namely, 2 mg. of hydrolysis of collagen1**a t bines onIy with the carboxyl groups of the collagen, as with copper, trypsin acting on a casein a hydrogen-ion concentrahydrolysis is as great as with untanned collagen, and does not depend substrate a t pH = 8 a t tion pH = 5.9, and a t on the amount of tanning agent present. Where the tanning agent, 40.00’ C. for half an hour. the temperature of 40” C . such as formaldehyde and quinone, combines with the amino groups Under these conditions 2 Since the chemistry of the of the collagen, the amount of hydrolysis depends on ( a ) the nature mg. of the enzyme gave 17.7 mechanism by which collaof the linkage-i. e., the type of tannage, and (b) the amount of tanmg: of soluble nitrogen. gen combines with various ning agent combined with the collagen. Chrome collagen is not This represents a trypsin substances to form leather is hydrolyzed by trypsin. strength approximately onestill almost entirely speculathird that of the strongest tive, it seemed possible that interesting results might be obtained by subjecting collagen, high-grade commercial preparation used by Sherman and tanned in various ways, to the action of trypsin. Not only Neun. As much as 20 mg. of the enzyme tested under the would this help in elucidating the theory of tanning, but it conditions requisite for pepsin activity gave no soluble nimight also give some idea of the mechanism of tryptic hy- trogen whatever. The acidity was controlled by a buffer solution, the hydrodrolysis. The primary difficulty in the study of the hydrolysis by gen-ion concentration being determined electrometrically. trypsin of collagen which has been treated with various METHOD tannrng agents lies in the fact that most of the substances used for tanning-e. g., heavy metal salts and formaldehydeFine siftings (100 mesh) of hide powder were tanned in are very definite enzyme poisons. Nevertheless] it seemed solutions of basic chromium sulfate, quinone, formaldehyde, a t least possible that by thorough washing of the treated copper sulfate, and gallotannin, washed and dried as dehide powder all soluble and ionized matter could be removed, scribed in detail below. About 0.5 gram of tanned hide leaving merely the insoluble combination of collagen and tan- powder was placed in a 10-cc. centrifuge tube with a conical ning agent. There then seemed no reason why a more bottom, graduated in tenths of a cubic centimeter. Ten complex compound, such as that of the collagen tanning agent, cubic centimeters of the buffer solution a t pH = 5.9 conshould not be hydrolyzed by trypsin, just as collagen alone taining 0.5 per cent trypsin were added. The tubes were is hydrolyzed. It is to be remembered that these various then corked and fastened to a shake machine, rotating at 8 tanned collagen compwnds are in general characterized by r. p. m. in a water thermostat a t 40.00’ C. After rotation the fact that, unlike collagen alone, they are not attacked by for 20 minutes, the tubes were removed and centrifuged for boiling water to yield gelatin, although some-e. g., vege- 20 minutes a t 1200 “times gravity.” Control tubes in which table-tanned collagen-are not entirely unchanged by hot the trypsin was omitted were run parallel with the digestions water. There is further the possibility that the trypsin and the percentage of hydrolysis determined by comparison might; hydrolyze the tanned collagen sufficiently to liberate of the volumes. enough of the tanning agent to inhibit the further action of The accuracy obtained in this method of measurement is the trypsin by “poisoning” it-i. e., presumably by combin- limited entirely by that in reading the level in the tubes. ing with or precipitating it. With the centrifuge and the fine sifted hide powder it was Yet the possibilities of throwing light on (a) the point of possible to obtain a well-defined boundary, and the percentage attack of the trypsin in the collagen molecule, and (b) the digestions so obtained are accurate to ~2 per cent. Connature of the combination in each tannage, seemed such that sidering the insoluble nature of the substrate, the method is they warranted the employment of this method of attack. probably the most accurate available, while being reasonably rapid. It is distinctly preferable to filtering off the undiMATERIALS USED Standard hide powder was chosen as the source of collagen. gested hide powder and determining that dissolved by an The trypsin was a high-grade commercial product which was estimation of nitrogen in the filtrate, a procedure which is objectionable and inaccurate for several reasons. Only 1 Received June 25, 1923. Presented before the Division of Leather small quantities of liquid are available. It is difficult to Chemistry a t the 66th Meeting of the American Chemical Society, Milfilter off the undigested hide powder satisfaCtorily and to waukee, Wis., September 10 t o 14, 1923. 9 Prom a part of the dissertation submitted b y Mr. Seymour-Jones in obtain a clear filtrate; the degradation products of hydrolypartla1 fulfilment of the requirements for the degree of doctor of philosophy sis are in part molecularly and in part colloidally dispersed, in the Faculty of Pure Science, Columbia University, June, 1923. Conand any filtration will merely effect an arbitrary separation tribution No. 431 from Columbia University. dependent upon the size of the filter pores. More particu* Numbers in text refer to bibliography a t end of article.
I
INDUSTRIAL A N D ENGINEERING CHEiMISTRY
158
larly, part of the nitrogenous matter in solution will be absorbed by the undigested hide powder, and a true aliquot will be impossible. CHROME-TANNED HIDEPOWDER
collagen was decomposed. Neither quinone nor hydroquinone appears to destroy the trypsin. The group a t which the collagen normally hydrolyzes with water appears to have been so changed by the tannage that hydrolysis no longer occurs. With trypsin some masking of the attacked group is shown by the lessened hydrolysis, which degree of hydrolysis varies with the amount of quinone used in tanningi. e., in combination with the collagen. Quinone tannage was discovered by Meunier' in 1908. His view3 of the process is that it takes place by oxidation of the collagen and reduction of the quinone. Representing the collagen as R-"2, we may have either
A chrome liquor of approximate composition Cr(0H)SOI was made up from chromium sulfate and sodium hydroxide to give 1.04 per cent chromium. Ten grams of 100-mesh h'ide powder were tanned in 200 cc. of this liquor ( A ) , and 10 grams further in 200 cc. of the stock liquor diluted 1: 4 with water-i. e., 0.21 per cent chromium ( E ) , for 22 hours on the shake machine. The tanned powders were thoroughly washed with distilled water until no test for Cr+++ R-NHI could be obtained, then were dried a t 75" C. Hydrolysis was then carried out as described above, the 2R-NHz results being given in Table I. TABLE I-DIQESTION
3.6 6.9
B
0.0 0.0
Further sets of hide powder were tanned in basic chromium sulfate solutions of various concentrations, but when the tanned powder was subsequently treated a t pH = 5.0, some of the chromium was stripped from the leather. Even with this, only the lightest tanned powder showed any digestion with trypsin. It was not found possible to prepare chrome liquors for tannage a t pH = 5.9, since the chromium began to precipitate out a$ this hydrogen-ion concentration. It appears from this that where hide powder is completely chrome-tanned, it is not hydrolyzed by trypsin. Whether this is due to the poisoning action of the chromium on the trypsin, or to the chromium masking the linkage which is attacked by trypsin, is not definitely established. I n any case, the theory of chrome tannage3 is a t present in so uncertain a condition that no light can yet be thrown on the action of trypsin in this particular reaction. QUINONE-TANNED HIDEPOWDER About 10 grams of 100-mesh hide powder were drummed up for 24 hours in each of three solutions of 1.0 (a), 0.5 ( b ) and 0.25 per cent (c) quinone, respectively, a t pH = 5.9, then allowed to stand for 48 hours, thoroughly washed to remove all uncombined quinone, and dried out a t 40" C. Hide powder tanned in a 0.1 per cent quinone solution was discarded because it was obviously undertanned. These tanned powders were analyzed, giving the results shown in Table IIa. QUINONE-TANNED LEATHERS Hide Substance Quinone ( N X 5.62) (by Difference) 84.1 7.3 4.5 86.7 89.6 1.2
TABLE IIQ-COMPOSITION Quinone Collaeen
OF
Water 8.6 8.8 9.2
Q
b c
Half-gram lots of these three tanned hide powders together with some untanned hide powder for comparison were digested with trypsin as described before. Subsequently, the residual liquors were poured off, fresh trypsin and buffer solution added, and a further digestion period of 20 minutes was given. TABLE II&DIGESTIONOF uinone ollaaen -
8
a b C
Raw collagen
QUINONE LEATHERS
PERCENT HYDROLYSIS1st 20 Minutes 2nd 20 Minutes Trvusin Control Trypsin Control .0 61 0
r-
268 :
0
86
13
0
69 79 91
4-2CeH4On
0 0
23
Considerable quantities of quinone and hydroquinone were liberated during hydrolysis, showing that the quinone
+ CsHe(0H)z
R-N/'>eH, ' 0 4- 3CsHaOz + R-N-0 +
1
R-N-0
OF CHROME LEATHERS
-PER c&mHYDROLYZEDTrypsin Control
Chromed Hide Powder A
Vol. 16, No. 2
(a)
+ 2CeHdOH)p ( b )
)CBHI
These reactions are exactly like those between quinone and aromatic amines. Support is lent to this view by the fact that hydroquinone is found in,the solution after tannage. Fahrion,6 on the other hand, presumes a preliminary oxidation of the collagen, followed by combination with the quinone. 2R-NHs R-NH-HN-R
+ H20 )C&
+ 0 R-NH-HN-R + CsH401+ R-"-0 +
R-NH-0
(a) (b)
This view does not explain the presence of hydroquinone in the residua1 liquor, unless it be supposed that the oxygen necessary for the first reaction is furnished by reduction of the quinone. Whichever view of the mechanism of the tannage is accepted, it is the amino group which is linked through oxygen to the benzene ring. The quinone-tanned collagen is readily hydrolyzable by trypsin, although not quite so rapidly as the untanned hide powder. The combination therefore exerts only a slight retarding effect.
FORMALDEHYDE-TANNED HIDEPOWDER About 10 grams of 100-mesh hide powder were drummed for 24 hours in each of four solutions of (a) 1.90, ( b ) 0.95, (c) 0.38, (d) 0.19 per cent formaldehyde a t pH = 5.9, then allowed to stand for 21 days, filtered, thoroughly washed, dried a t 40" C., and powdered. Half-gram lots of these tanned hide powders were digested with trypsin, as described before. TABLE 111-COMPOSITION AND DIGESTION
OF FORMALDEHYDS LEATHERS Hide Formaldehyde water Substance (by Difference) P E R CENT HYDROLYSIS Sample Per cent Per cent Per cent Trypsin Control 7.1 87.8 6.1 6 5 88.8 4.2 8 14 6.4 7*0 90.3 3.3 18 3 8.2 89.0 2.8 35 0 . Raw collagen 86 13
.
f
f
..
..
..
The results shown in Table 111, particularly in the controls, are somewhat irregular. Formaldehyde was found to be present in all the residual liquors after digestion, and its destructive action on the trypsin may well account for the comparatively small hydrolysis in the more heavily tanned powders. Tannage with formaldehyde is of the type most generally acknowledged to be purely chemical in nature. It is usually represented as or R < N y COOH
coo
.
