44 SUBCELLULAR PARTICLES 



was through this experimental study, to be discussed below, that development 

 of the matrix concept became possible. 



Formation of Lignin in Matrix Systems. Cellulose in the form of analytical 

 grade filter paper, a tentative cell wall model, was incubated with eugenol, 

 peroxide and peroxidase. Within 20 hours, detectable amounts of lignin-like 

 product were found in the paper, but not in the ambient medium; approximately 

 I milligram per gram of paper was formed. Although this finding tended to con- 

 firm the postulated role for cell wall substance in polymer formation, the low 

 yield rendered the system nearly impracticable for extensive and critical experi- 

 mentation. It was recalled, however, that the peroxidase in the cell wall was 

 tightly bound, hence that a filter paper system with peroxidase in solution failed 

 to duplicate a wall condition of possible importance. Paper was accordingly 

 infiltrated with peroxidase solution (2X 10^*^ moles/gm) and dried under reduced 

 pressure at room temperature (32) or dried under normal atmospheric conditions 

 at o°C (j,^). It was correctly surmised that the enzyme might be sufficiently 

 strongly adsorbed on the cellulose to render its elution slower and more difficult 

 when returned to an aqueous environment. The peroxidase-paper in a eugenol- 

 peroxide solution formed 12—25 milligrams of lignin per gram of paper, amounts 

 of product that rendered further experimentation feasible. The product formed, 

 in part soluble in dioxane, in part in ethanol-HCl, possessed the characteristic 

 ultraviolet absorption spectrum of a gymnosperm lignin (see fig. 2). The dioxane- 

 soluble fraction on analysis yielded C, 63-64%; H, 6-7^^; OCH3, 14-16%, 

 typical values, and gave positive color tests for lignin. 



In addition to a number of variations on the basic experimental design, in- 

 cluding grade and area of paper, efforts were made to introduce a chemical 

 block into the cellulose matrix. Paper acetylated sufficiently to block 25 per cent 

 of the cellulose hydroxyl groups yielded 53-69 per cent less lignin than untreated 

 paper {^^), and when increased to a 34 per cent block, yielded 73—90 per cent less 

 product. Thus, even when most of the cellulosic OH is free, lignin polymer 

 formation is greatly inhibited, suggesting that more than one OH is involved 

 in the orientation of each monomer, and evoking a picture of monomer units 

 forming a continuous file along the polysaccharide chain prior to polymeriza- 

 tion. An unexpected additional evaluation of the acetyl block was obtained when 

 chitin from the egg case of Busycon was tested as a matrix. Because it is a 

 2-amino-jS-^-glucopyranoside derivative, hence an analog of cellulose with 

 similar molecular and crystallographic properties ( 18), chitin provided an ex- 

 ceptionally useful material for comparison with cellulose; it was found to yield 

 about 5 milligrams of lignin per gram of matrix. In the natural state, the 2-amino- 

 group of chitin is acetylated and, assuming the H-bond donating amino group 

 to be equivalent to a hydroxyl group, native chitin should correspond to the 

 cellulose with a 34 per cent block described above. Upon deacetylation of chitin 



