Action of Trypsin on the Properties of Collagen1 - American Chemical


removal was the primary function of trypsin*in bating, except in those few cases where the process is prolonged sufficiently to cause partial digestio...
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1 S D CSTRIAL A X D E,VGIAYEERINGCHEMISTRY

May, 1930

dephlegmator to the washer, F , in which a caustic solution eliminates any trace of phosgene, chlorine, or any derivative of pentavalent antimony. From there the gases pass through a regulating valve to the gasometer, G, and thence to pump H , which ddivers the liquefied product to receivers located on a lower level. The operation is as follow: 125 pounds of sublimed antimony trifluoride are delivered to the autoclave through B , and the required amount of antimony pentachloride is added. Hole B is closed, the carbon tetrachloride wpply pump, I ,

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is started, and steam is turned on. The reaction starts immediately and in a short while the pressure reaches 60 pounds; the subsequent operation is automatic. The end point is very sharp and corresponds to the theoretical quantity of tetrachloride. The pressure is then released through a by-pass valve on the pressure regulating system. The dump valve is opened and the molten antimony trichloride flows by gravity iuto hooded containers. N o further cleaning is required. After the dump valve is closed the apparatus is ready for the next run.

Action of Trypsin on the Properties of Collagen' R. 0. Page and A. W. Page Jl

OOLSTO\

T%IZ\ERIES, \ ~ ~ O O I . S T O N ,

I S C E the pioneer work of Wood ( I S ) on the niechanisni (if bating, the functions of the various constituents of the hate have been widely studied, more especially by Kilson and Jlerrill, who have paid a great deal of attention to the action of the enzyme. In 1920 Wilson showed that in bating elastin was attacked by trypsin (8) and the following year he carried the study of elastin removal further ( 1 0 ) . setting forth the factors controlling it and Phowing that it was the only action of a bate liquor observable under a microscope. As a result of this rT-ork it was generally assumed that elastin removal was the primary function of trypsiain bating, except in those few cases where the process is prolonged sufficiently to cause partial digestion of the collagen itself. A few years later, however, Kilson and Merrill ( I I ) showed that not only was keratose (formed by the action of lime solutioas on keratin) also removed by bating, but it' was attacked much more rapidly than elastin, and indeed in the case of certain types of calfskin all measurable effects of bating were produced before any noticeable action on the elastin had started. .Isit was found possible to correlate the keratose-digesting power of a n enzyme with its bating value, it seemed very probable that in working with calfskins this Tvas the important function of the enzyme. Jfarriott (3) attacked the theory that the essential function of the enzyme was the removal of elastin by drawing attention to the fact that enzyme-treated skins, when tanned, are always softer and lighter in color than skins treated similarly, except for the absence of enzyme. He attributed the difference to the removal by the enzyme of degraded or $-collagen while the unaltered or a-collagen remained unattacked, and claimed that this reinoval of degraded collagen, t,ogether with the cleansing of the grain, presumably by the solution of keratose, is the essential change caused by the enzyme during the bating proc This viewpoint centers attention on the action of the enzyme on the collagen, and in this connection Merrill (5, 6) has shown that collagen is attacked by trypsin at exactly the same rate whether from limed or unlinied calfskins, and that during the normal bating of calfskins only negligible traces of collagen are removed. While this Jvork indicates t,hat hlarriott's assumption of the preferential solution of degraded collagen is untenable, the mere removal of keratose seems insufficient to account for the appreciable effect which bating sometimes has on both the firmness and color of the resultant leather. Although it is widely recognized that bating tends to produce softness in leat'lier and t,hat this softness is often connected with the falling which takes place in that process,

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' Received

December 23, 1929.

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JIerrill ( d ) , in his study of the rat'e of falling of calfskin during bating, found that this was increased by enzymes only when present in quantities much greater than any el-er employed in practice, and ascribed the increases then observed to the actual destruction of a considerable portion of the skin. Previous work in this laboratory (?), hoiverer, demonstrated that hide plumps in lime progressively as the time is increased. n-hile Gust'avson ( 2 ) has shown that increase in time of liming increases the power of hide to combine wit'h tanning materials. Moreover, the plumping due to long liming cannot be entirely reversed by mere reduction-of the p H value to the isoelectric point of collagen. The present paper is a continuation of this line of investigation. Effect of Pretreatment of Collagen on Enzyme Action

Portions froin the butt of a cowhide were soaked for 23 hours in each of two changes of water and then limed for 5 days in saturated h i e solution cont'aining excess of lime and 0.1 nornial in sodium sulfide. After being unhaired, . t h e pieces were washed for 2 hours in running w t e r , delimed by means of ammoniuin chloride, and then bated for 24 hours at 35" C. in a solution containing 0.2 gram of trypsin per liter and 0.1 niolar in sodium phosphate adjusted to p H 8. On removal from the bate, the pieces were washed in running water for 3 hours. Sole-It will be noted t h a t this time period of bating is f a r in excess of what would be used in practical tannery operation with this class of hide. T h e results obtained, therefore, should not be construed a s indicative of what happens in practice but are, rather, of theoretical interest.

It was considered that this treatment would remove all the elastin present. That keratose and other readily soluble protein material were also removed was also shown by the fact that upon further treatment with the enzyme a slow, uniform solution of nit'rogen took place. It seemed safe t'o assume, therefore, that the hide pieces a t this stage represented collagen fairly free from other proteins. Their thickness having been measured by means of a gage pressing on them wit'h uniform pressure for 1 minute, the purified pieces were kept for 3 days in a larger reservoir containing 0.05 A' sodium hydroxide solution, then neutralized with a n equivalent amount of hydrochloric acid, complete penetration of which was insured by constant shaking, and finally washed thoroughly in water. The pieces were next placed in 0.1 M sodium phosphate solutions of p H 8 containing different amounts of enzyme and maintained a t 35' C. for 24 hours. They were then removed

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