Frozen Collagen 



111 



and almost complete destruction. There are, however, 

 differences in the behaviour of the periodic structure 

 of the fibrils. When treated with papain this charac- 

 teristic structure is preserved when the damage is 

 slight or of moderate severity. It nearly always 

 disappears when the damage is severe. But in fibrils 

 treated with acid solutions it persists even in the 

 zones of severest damage. In fibrils treated with 

 moist heat above 60 C the periodic structure always 

 disappears by a transformation into collagenous 

 gelatin, regardless of the degree of fibrillar damage. 

 The changes produced by papain and papaiotin 

 are morphologically similar but not identical with 

 those produced by other factors. These changes arc 

 as much proof of the proteolytic action of the fer- 

 ment as is the formation of amino acids from the 

 chemical point of view. It thus seems possible that a 

 technically perfected morphological examination 



may one day funiisli us with a simple method for an 

 approximate assay of the proteolytic action of some 

 ferments. 



References 



M. E., Kciul. ist. super. Siiniia 10, 517 



1. Alessandrini, 



(1947). 



2. Hall, C. E., Jakus, M. A., and S( iimitt, F. O., /. Amer. 



Clu-m. Soc. 64, 1234 (1942). 



3. Lelli, G., Jornada med. 5, 397 (1952). 



4. Lelli, G. and Marotta, U., Rend. ist. super. Sanita 13, 



518 (1950). 



5. RoHH-SMirii, A. H. T., Significance orCollagcnasc. Nature 



and Structure of Collagen. Butterworihs Scicnt. Pub!. 

 London, 1953. 



6. Rudall, K. M. (1952) quoted by Robb-Smith, see ref. (5). 



7. Willsfalttlr and Grassmann, Z. plivsiol. Cliem. 138, 



184(1924). 



8. WoLPERS, C, Klin. Wochensihr. 22, 624 (1943). 



9. Wyckoff, Electron Microscopy. Intcrscience Publ. New 



York, 1949. 



Electron Microscopic Observations on Frozen Collagen 



G. Lelli and G. Arangio-Ruiz 



Istitiito Siiperiorc di Sanita. Roma 



It is known that the fibrils of collagen are particu- 

 larly sensitive to variations in pH so that even weak 

 acid solutions can seriously damage them. When 

 such damage is slight, it consists of a flattening and 

 enlargement of the fibrils, when it is severe it pro- 

 duces swelling which may be total or partial up to 

 the complete dissolution of the fibrils in the liquid 

 medium. 



The varying degree of damage is generally related 

 to the duration of contact with the acid solution, its 

 temperature and the quality and age of the collagen. 



In the course of electron microscopic studies on 

 this subject we happened to notice that collagen 

 preserved at low temperatures sometimes showed a 

 greater resistance to the disintegrating action of acid 

 solutions. 



The material used in these experiments was collagen 

 from guinea pig skin and rabbit or calf tendon. 



In a first group of experiments carried out on 20 

 samples of collagen, half of each sample was frozen in a 

 refrigerator at — 70 C for a period ranging from 20 to 

 144 hours. It was then lacerated in water, treated with 

 a lactic acid solution of a pH 3.5 for 20 minutes, then 

 washed in water and examined electron microscopically. 



The other half of each sample was used as a control 

 and treated in exactly the same way without previous 

 freezing. 



The action of the acid solution was always visible 

 electron microscopically and showed the usual fibril- 

 lar damage. When the increased resistance of the 

 frozen hbrils, as compared with the controls, was so 

 great as to produce a marked difference in the 

 number and severity of the lesions (fig. I) this 

 was recorded as a positive result. This criterion. 



although admittedly of only approximative value, 

 has none the less yielded some interesting data on 

 the existence of the phenomenon. The number of posi- 

 tive readings show that in at least <S0 "„ of the cases 

 the collagen fibrils of frozen tissue offer more and 

 even double the resistance to acid solutions as 

 compared with non-frozen material. 



In a small number of tests (5) the lactic acid was 

 replaced by a 2 "o solution of papain for 24 hours 

 at 30 C. 



The results as judged by the same criteria as were 

 used in the case of the acid solution, also showed 

 the greater resistance of frozen collagen fibrils to the 

 disintegrating action of the proteolytic cnz>me. 



HM<<t«>^ > 



^:. ^ 





Fig. 1. Damage of the structural organization of collagen 

 fibrils produced by treatment in acid solution. 



