59 w ithin the experimental error When the concentration of

solvent wa© really critical, e.g. in the meaGuremente of initial drop in viocosity on heating as a function of KI concentration, the filtered protein solution was dialyeed against solvent of the exact final concentration.

The viscosities of KÎ and CaClp solutions per so were corrected for by plotting the draining times of the pure

salt solutions as a function of concentration and subtracting the appropriate values from the draining times of @alt-protein solutions. Temperature was controlled at 20^C. with a

water bath.

The protein content of the solutions was calculated from nitrogen estimation.

10. Estimation of proteolytic activity.

Enzyme studio© were carried out during this work for the following purposes

(a) to determine whether proteinase© would degrade collagen in a medium of concentrated salt.

(h) to provide a relative measure of the resistance to degradation of diversely prepared collagen fibres.

For the first purpose a variety of enzymes was used, namely, trypsin, collagonaso, papain, hyaluronidase, pronase, and fungal protease. Of those, trypsin was the crystalline

commercial preparation and the rest were commercial powders.

The drop in the viscosity of a collagen solution in salt was used as an index of their activity, with an enzymeîsubstrate

ratio of 1:10. A weighed amount of the solid enzyme was added to 20 ml, collagen solution of known concentration

(approximately 0,1%) such that this ratio was obtained, and the specific reduced viscosity of this system compared with that of the original. The equilibrium condition was allowed to occur before the final reading. The viscometric technique is treated in detail in Section II, 9, (ii).

For (b) above, the amount of nitrogen released from

insoluble collagen fibres after papain treatment was measured. Crude papain is a mixture of enzymes (Smith and Kimmel, 1957). Papain itself has a very wide specificity (Smith and Kimmel, 1957) and so relative resistance to degradation by this

enzyme system should be a good index of the relative stability of collagen fibres, 1 ml. papain solution (5% in O. DIM

cysteine) was added to fibres prepared as in Section II,4(ii), After set times the residual fibre© were filtered off on mats of cotton wool and the filtrate estimated for nitrogen

(Section II, 5). Correction was made for the contribution of the papain and cysteine to the nitrogen value and the amount of nitrogen brought into solution calculated as a percentage of the original total nitrogen.

6 1

il. Solubility studies.

The measurement of the solubility of a protein which becomes more soluble on dénaturation presents something of a problem since at normal temperatures it will be subject to a slow thermal dénaturation and the solubility will be partially a function of time. This can be obviated to some extent by allowing the solubilisation to take place in the cold room, but it ia obviously essential to allow exactly the same lengths of time for treatments which are to be compared, The major difficulty is then to prepare the starting material, i.e. insoluble collagen, in such a way that the particles have the same total surface area in each aliquot treated. The first method attempted (to find the relative solubility of collagen in salts as a function of pH) was to weigh out equal portions of freeze-dried collagen and shake these with the appropriate solution for 48 hours. Consistent results could not be obtained, almost certainly due to different surface areas of the sponge-like particles. In an attempt to obviate this, 10 ml. aliquots of an acetic acid solution of collagen of known concentration (0.25%) were run from a burette into segments of Visking tubing and the protein precipitated by dialysis against distilled water for 7 days. The sacs were then placed in 1 litre of the

and Xef?t for tea day© in the cold room (9^C) with occasional manual agitation. At the end of this time, the sacs were washed out with a small volume of distilled water,

centrifuged, and the nitrogen content of the precipitate and supernatant measured* This method gave reasonably consistent results but is still not entirely satisfactory since (a) it is difficult to ensure that all the sacs are the same shape on dialysis, which is necessary if the same surface area of precipitate is to be obtained, and (b) it is difficult to ensure that all the material in the sacs has been removed at the end* Moreover the technique is extremely cumbersome*

Later, a technique similar to that described by Gallop et al» (19S7) was employed* Collagen in the form of the desalted gel was homogenised with a little distilled water to try to achieve a uniform suspension of small collagen particles, 2 ml* aliquots of this were pipetted into centrifuge tubes, and 2 ml. of the required salt solution added* After shaking for 6 hours, the residual suspension was centrifuged down, washed with two 2 ml* portions of the same solvent, and analysed for nitrogen. Subtraction of this value from the total initial nitrogen present yielded the percentage solubilised* This gave consistent results and is now the method of choice, although it is open to the

63