LIGHT AND THE PIGMENT 107 



a tissue which was more or less colorless. Corn seedHngs do not 

 synthesize chlorophyll when they are grown in the dark but do 

 respond to red and far-red light. Such organisms should provide an 

 excellent source of plant material for isolation of phytochrome. 

 Obviously, since the seedlings appear colorless, the pigment must be 

 very dilute. 



Since the pigment is so dilute, the primary problem is to be able 

 to measure it with sensitive instruments. The spectrophotometer 

 which finally matched the requirements was originally built to measure 

 very slight changes in the light intensity of a light beam passed through 

 a very dense sample of material. Intensity changes amounting to a 

 small fraction of 1 % could be measured in a beam of light passed 

 through 5 cm (2 in.) of wood! Thus a great many corn seedlings 

 could be packed together in a relatively large container, and light of 

 any wavelength which was absorbed by these seedlings could be 

 measured with a high degree of accuracy. 



Next came the problem of learning how to recognize the pigment 

 with this instrument. The solution was to illuminate the sample with 

 high intensity red or far-red light and then immediately to measure 

 the difference in absorption of red and far-red. The source of red 

 and far-red for measuring the absorption alternated about 16 times 

 each second. Pre-illumination with red should result in high 

 absorption of far-red compared to red and vice-versa. 



Early tests with the corn seedlings demonstrated that phytochrome 

 could be recognized with the instrument. Then corn seedling tissue 

 could be fractionated in various ways, and the most active fractions 

 could be determined. Following this procedure, phytochrome, which 

 proved to be a protein with a molecular weight of about 200,000, was 

 extracted first in a concentration of about 10"^ moles per liter. It 

 has been concentrated and purified by special column chromato- 

 graphy to the point where it is about 3 % pure in a pellet of protein. 

 In this form and in water suspension it is possible to see the bluish- 

 green color and the slight color change that occurs upon illumination 

 with red followed by far-red light. By the summer of 1962 the 

 pigment had also been extracted from green plants. It is interesting 

 that pigment could be obtained from a number of long-day plants 

 but not from the short-day plants which were tried, including 

 cocklebur. It is obviously also present in short-day plants, and the 



