172 W. M. McKernan, S. D. Dailly 



Experimental 



Raw material 



The inside portion of dehaired, defleshed bovine hide was intensively washed in 

 distilled water to remove all traces of lime salts and was blotted dry. 



Bovine bone collagen was isolated from fresh cattle bones which were thoroughly 

 degreased with water (Chayen and Ashworth, 1953) and the mineral matter 

 removed by extensive demineralisation in N hydrochloric acid with several changes 

 of acid. This treatment was continued until a constant value of phosphorus content 

 was obtained for the bone collagen. Residual acid was removed by thorough washing 

 and the collagen was blotted dry. 



Swelling curves 



Swelling curves were determined over a relatively short time period at pH 2.3 

 (the maximum acid swelling pH for corium collagen) and at pH approx. 14.0. Bowes 

 and Kenten (1950 a) have shown that the swelling of collagen in sodium hydroxide 

 solution increases progressively with increase in pH and shows no pH maximum 

 corresponding to that occurring in acid solution. 



10 g. samples of the purified collagens were suspended in 50 ml. 1.0 N sodium 

 hydroxide or hydrochloric acid solution (pH 2.3) for varying periods of time up to 

 24 hours at room temperature. At various intervals samples were removed and 

 weighed. In all experiments swelling was taken as the amount of solution retained 

 expressed as a percentage of the weight of the original collagen. 



Phosphorus 

 After determination of the swelling factor the collagen was washed and dried 

 overnight at 105 ^C and a known weight ashed at 550 '"C. Phosphorus was deter- 

 mined as the molybdovanado-phosphate complex using a slight modification of the 

 method of Michelson (1957). Potassium dihydrogen phosphate was used for the 

 determination of the calibration curve. 



Results and discussion 



The effects of time on the swelling of collagen in solutions of sodium hydroxide 

 and hydrochloric acid are shown in Fig. 1 and Fig. 2 for bone collagen and corium 

 collagen respectively. In the series of experiments carried out in acid solution the 

 curves obtained for purified bone collagen are similar to those described by Veis and 

 ScHLUETER (1964) for dentine collagen and confirm the lack of swelling properties of 

 this type of hard tissue. Extension of the swelling time from 24 hours to several days 

 did not result in any increase in the swelling factor. 



In contrast, in alkali, bone collagen behaves similarly to corium collagen, ex- 

 hibiting a rapid uptake of solution during the 24 hour period. Extension of the 

 swelling period beyond this time leads to rapid disintegration of the collagen. 



A series of experiments was also carried out whereby purified bone collagen was 

 removed from alkali after a swelling period of 9 hours, thoroughly washed and 

 re-suspended in hydrochloric acid at pH 2.3. Further swelling investigations were 

 carried out at this pH as shown in Fig. 1. No further increase in swelling was ob- 

 served although the phosphorus content of the bone collagen had been reduced by 

 approximately 80", o during the 9 hour alkaline treatment (Fig. 3). The series of 



