COMBINED FORMS— EXTRACTION 33 



By combination of the protein from one flavoprotein with the prosthetic 

 group from another, new flavoprotein enzymes can be formed. 34 Some of 

 the flavoproteins are thought to have prosthetic groups in addition to the 

 flavin nucleotides. 44 



In view of the fact that riboflavin appears always to be attached to 

 proteins through a phosphate radical, it is not surprising that its extrac- 

 tion from tissues has not generally been a serious problem. Snell and 

 Strong 46 recommend for quantitative extraction autoclaving in the pres- 

 ence of a large amount of water. Extraction with O.liV acid is also men- 

 tioned by these authors and has been widely used. Neither procedure 

 effects a quantitative extraction from all materials, even though the 

 extraction may often be complete or nearly so. 8 Recently the use of 

 0.25A r HC1 in 25 per cent acetone and 75 per cent water has been recom- 

 mended. 47 The U.S. Pharmacopoeia method involves autoclaving at 15 

 lbs. pressure for 30 minutes with 0.04./V HC1. 



Digestion with a phosphatase preparation to obtain free riboflavin has 

 theoretical justifications because of the existence of riboflavin bound to 

 phosphoric acid, and the lack of definite information with respect to the 

 hydrolysis of this combination under acid conditions. Alkaline hydrolysis 

 cannot be used because riboflavin is unstable under alkaline conditions. 



Recent experimental studies have shown that the cooking (and storage) 

 of various foods causes a considerable increase in their assayable ribo- 

 flavin content. This extra amount of riboflavin appears not to be released 

 by phosphatase preparations, and to be determinable both by fluorometric 

 and microbiological methods. 48, 40 



Nicotinic Acid, Nicotinamide 



Nicotinic acid occurs principally in the form of its amide, and in tissues 

 this is for the most part linked directly through the pyridine nitrogen to 

 a ribose residue which makes up a part of a dinucleotide, such as co- 

 enzyme I: nicotinamide-ribose-phosphate-phosphate-ribose-adenine. This 

 coenzyme is in turn linked to various proteins (apoenzymes). Coenzyme 

 II has a structure similar to coenzyme I except that there is another 

 phosphate radical the position of which is uncertain. Nicotinamide is 

 linked in the same manner in both coenzyme I and coenzyme II, either of 

 which in turn may be combined with a number of proteins. Sumner and 

 Somers describe about 15 dehydrogenases of which about twice as many 

 contain coenzyme I as contain coenzyme II. 50 The isolation of dinicotinyl 

 ornithine from the excreta of chicks 51 shows that in natural materials 

 nicotinic acid may be combined through an amide linkage to other 

 structures. 



