ANALYTICAL BIOCHEMISTRY
70, 218-223 (1976)
A Sensitive Assay for Collagenolytic Activity Using Tritiated Collagen KINGSLEY R. LABROSSE. IRVIN E. LIENER. AND PAUL A. HARGRAVE
Depa rtment of Biochemistry, College of Biological dence , University of Minnesota, Sf. Paul, Minn esota 55108, and Department 0/ Chemistry alld Biochemistry, Southern Illinois Univer it " Carbondale', lIIinoi.1 62901
Received April 30. 1975; accepted
ugu t 20, 1975
A method i described for the a ay of collagenolytic activity bed on the usc of collagen which has been tritiated by the free-rawcallOterceptor method. he high speci1ic activity of the radioactive collagen aJlow for reater en itivityand horter time of incubation than pre ently available assay for collagenolytic enzy mes. The tritiated collagen appears to have underaone little change in its n live tructure and is refractory to proteolytic attack by tryp in, chymotrypsin, and elastn e. Thi assay affords a sensitive mean of measuring the true collagena e a livity of mammalian tumor tissue .
Assays presently available for mea uring collagenolytic enzyme generally involve the use of large amount of collagen a the ub ' trate and long periods of incubation . For example, the vi co· ity method de cribed by Seifter and GalJup (1) require a final concentration of collagen in each assay system of 8 mglml. The a' ay ba ed on the relea e Ofl'4 ]glycinecontaining peptides from He-labeled collagen (2) not only nece sitate the laborious isolation of thi ub trate from animal that have been injected with (l4C]g1ycine, but al 0 require incu bat i n period of up to 18 hr in order to obtain meaningful re ults . Other method include the procedure of Mandl et al. (3) wherein native in oluble collagen i u ed a the substrate, and the acid- oluble degradation produc are determined by assaying for free amino group with ninhydrin (4) or for hydr xyproline (5). These latter method , in particular, uffer from lack of en itivity which is frequently an overriding con ideration in a aying crude ti ue extracts for collagenolytic activity. In this paper we describe a imple. en itive radioa tive as ay for collagenolytic activity which involve the ue of [3H] coli gen which may not only be employed for creening crude ti sue extract for c lIagen lytic enzymes but also for more preci e kinetic tudie . The technique u ed for the tritiation of collagen cau e minimal damag t the native collagen. which is an important consideration becau e of the high degr e of specificity whjch collagenase are known to exhi it t ward native collagen in soluble or fibrillar form (6). 21 Copyriaht
C
J976 by AcademIC Pre , I II
70, 218-223 (1976)
A Sensitive Assay for Collagenolytic Activity Using Tritiated Collagen KINGSLEY R. LABROSSE. IRVIN E. LIENER. AND PAUL A. HARGRAVE
Depa rtment of Biochemistry, College of Biological dence , University of Minnesota, Sf. Paul, Minn esota 55108, and Department 0/ Chemistry alld Biochemistry, Southern Illinois Univer it " Carbondale', lIIinoi.1 62901
Received April 30. 1975; accepted
ugu t 20, 1975
A method i described for the a ay of collagenolytic activity bed on the usc of collagen which has been tritiated by the free-rawcallOterceptor method. he high speci1ic activity of the radioactive collagen aJlow for reater en itivityand horter time of incubation than pre ently available assay for collagenolytic enzy mes. The tritiated collagen appears to have underaone little change in its n live tructure and is refractory to proteolytic attack by tryp in, chymotrypsin, and elastn e. Thi assay affords a sensitive mean of measuring the true collagena e a livity of mammalian tumor tissue .
Assays presently available for mea uring collagenolytic enzyme generally involve the use of large amount of collagen a the ub ' trate and long periods of incubation . For example, the vi co· ity method de cribed by Seifter and GalJup (1) require a final concentration of collagen in each assay system of 8 mglml. The a' ay ba ed on the relea e Ofl'4 ]glycinecontaining peptides from He-labeled collagen (2) not only nece sitate the laborious isolation of thi ub trate from animal that have been injected with (l4C]g1ycine, but al 0 require incu bat i n period of up to 18 hr in order to obtain meaningful re ults . Other method include the procedure of Mandl et al. (3) wherein native in oluble collagen i u ed a the substrate, and the acid- oluble degradation produc are determined by assaying for free amino group with ninhydrin (4) or for hydr xyproline (5). These latter method , in particular, uffer from lack of en itivity which is frequently an overriding con ideration in a aying crude ti ue extracts for collagenolytic activity. In this paper we describe a imple. en itive radioa tive as ay for collagenolytic activity which involve the ue of [3H] coli gen which may not only be employed for creening crude ti sue extract for c lIagen lytic enzymes but also for more preci e kinetic tudie . The technique u ed for the tritiation of collagen cau e minimal damag t the native collagen. which is an important consideration becau e of the high degr e of specificity whjch collagenase are known to exhi it t ward native collagen in soluble or fibrillar form (6). 21 Copyriaht
C
J976 by AcademIC Pre , I II
