106 THE PHYSIOLOGY OF EARTHWORMS 



charge of oxygen is 0-070 sec at 23 °C and pH 8 (Salomon, 

 1941). 



Information gained from centrifugation and from the spectral 

 analysis leads to the conclusion that one type of haemoglobin is 

 present. The technique of alkaline denaturation, however, can be 

 used to determine whether one or more types, having essentially 

 similar properties, are to be found. Blood obtained fresh and 

 brought to an alkaline pH (10-5-13) is converted from oxy-haemo- 

 globin to alkaline globin haemochromogen. Under controlled 

 conditions this reaction can be followed by monitoring the 

 proportion of unchanged oxyhaemoglobin in solution. 



The time taken for the reaction to go to completion differs from 

 species to species, and from pH to pH. At pH 12-7 complete 

 denaturation takes 23 minutes for L. terrestrishut only 6 minutes for 

 A. longa. L. festivus 11-5 minutes, E. foetida 5 minutes and L. 

 ruhellus 2-5 minutes, all show individual denaturation times 

 (Haughton, Kerkut and Munday, 1958). A denaturation time of 

 about 24 minutes is given by Manwell (1959) for L. terrestris 

 haemoglobin at pH 11-7. Denaturation proceeds more slowly in less 

 concentrated alkali, taking about 34 minutes at pH 11-2 and 50 

 minutes at pH 10-92 (Manwell, 1959). 



The construction of curves showing the course of denaturation 

 reveals that the process is not a simple linear event, but is composed 

 of a number of phases. According to Haughton, Kerkut and 

 Munday (1958) the process takes place in two stages, the initial 

 stages of denaturation proceeding more quickly than the later 

 stages. The slope of the curve is steep and straight until the 

 concentration of unchanged oxyhaemoglobin is only 30% and then 

 the line breaks to give a different more gentle slope until completely 

 denatured (Fig. 33). Arguing from the different denaturation 

 times of mammalian adult and foetal haemoglobins (Brinkman 

 and Jonxis, 1937) it is thought that the two portions of the curve 

 are indicative of two forms of haemoglobin in earthworms. Very 

 few points are given for the initial portion of the process, however, 

 and more information is given by Manw^ell (1959). He finds 

 evidence that the denaturation process is triphasic rather than 

 biphasic. Three equal stages occur, each accounting for about a 

 third of the haemoglobin. The form of the denaturation is constant 

 whatever the pH used. Instead of postulating three types of 



