Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The chemical (non-biological) and photolytic transformations of pteridines and purines effected by the… Landymore, Arthur Frederick 1971

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1972_A6_7 L35.pdf [ 7.58MB ]
Metadata
JSON: 831-1.0101496.json
JSON-LD: 831-1.0101496-ld.json
RDF/XML (Pretty): 831-1.0101496-rdf.xml
RDF/JSON: 831-1.0101496-rdf.json
Turtle: 831-1.0101496-turtle.txt
N-Triples: 831-1.0101496-rdf-ntriples.txt
Original Record: 831-1.0101496-source.json
Full Text
831-1.0101496-fulltext.txt
Citation
831-1.0101496.ris

Full Text

THE CHEMICAL (NON-BIOLOGICAL) AMD PHOTOLYTIC TRANSFORMATIONS OF PTERIDINES AND PURINES EFFECTED BY THE SALTS OF SEAWATER, AND THEIR ECOLOGICAL SIGNIFICANCE. by ARTHUR FREDERICK LANDYMORE B.Sc., University of B r i t i s h Columbia (1968) A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1971 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requi rements f o r an.advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re fe rence and Study. I f u r t h e r agree tha t permiss ion f o r ex tens ive copying o f t h i s t hes i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department or by h is r e p r e s e n t a t i v e s . I t is understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l lowed w i t h o u t my w r i t t e n pe rm iss ion . Department o f ABSTRACT. The degree of chemical i n s t a b i l i t y of pteridines (related to xanthopterin) and purines (related to u r i c acid) i n seawater was studied with a view ( i ) to assess i t s r o l e i n the ecological turnover of these compounds i n the marine environment, ( i i ) to define the i n t e g r i t y with which they may serve as nitrogen-source for growth of marine phytoplankters. Solutions of these compounds were incubated a s e p t i c a l l y at 2 0 - 2 5 ° C with i l l u m i n a t i o n from cool-white fluorescent lamps or i n complete darkness and the chemical changes were monitored spectrophotometrically. Among the purines tested, u r i c acid showed slow degradation i n darkness which was accelerated by l i g h t , while xanthine was degraded only by l i g h t . Adenine, guanine and hypoxanthine appeared to be stable. The pteridines tested included p t e r i n (2-amino-4-hydroxy-p t e r i d i n e ) , lumazine ( 2 , 4-dihydroxypteridine), and t h e i r following hydroxylated d e r i v a t i v e s : 6-monohydroxyl (xanthopterin, oxylumazine), 7-monohydroxyl (is©xanthopterin), 6 ,7-dihydroxyl (leucopterin, dioxylumazine). In general, they showed the following order of chemical s t a b i l i t y i n seawater: 6,7-unsub-s t i t u t e d > 7-monohydroxyl> 6,7-dihydroxyl> 6-monohydroxyl. The studies were extended to investigate whether the i n s t a b i l i t y was due to the pH or the s a l t composition of seawater and pertinent aspects of the underlying chemistry were explored. In darkness, p t e r i n , lumazine, and isoxanthopterin were completely sta b l e , whilst the other pteridines showed increasing i n s t a b i l i t y i n the order shown above. Excepting oxylumazine, a l l the pteridines showed chemical r e a c t i v i t y i n seawater a t t r i b u t e d to i t s pH and not i t s s a l t content. On the other hand, oxylumazine showed marked l a b i l i t y i n seawater a t t r i b u t a b l e to i t s s a l t content and not i t s pH. This p t e r i d i n e required minimal concentrations of s a l t and d iva lent trace-meta l ions 2 + (such as Cu ) to show the chemical r e a c t i v i t y observed i n sea-water. When the s a l t present was NaCl only, oxylumazine showed 1:1 ox idat ive conversion to dioxylumazine, but with the t o t a l sa l t s of seawater the conversion was 2:1 with h a l f of the oxylumazine being degraded, apparent ly non-ox ida t ive ly , to u n i d e n t i f i e d non-pter id ine products; t h i s l a t t e r degradation i s a t t r i b u t e d to the combination of anions present i n seawater. Unl ike oxylumazine, xanthopterin showed 1:1 ox idat ive degradation v i a l eucopter in i n seawater. In the l i g h t , a l l the p ter id ines showed greater i n s t a b i l i t y than i n darkness but with the same order of inf luence of subst i tuents on t h e i r r e a c t i v i t y . Excepting l eucopter in and dioxylumazine, the p h o t o l y t i c r e a c t i v i t y i n seawater was a t t r i b u t a b l e to i t s pH and not i t s s a l t content; th i s was a l so the case with oxylumazine which had shown anomalous behaviour i n darkness. Leucopter in and dioxylumazine (both 6,7-dihydroxy-la ted p t e r i d i n e s ) gave evidence of r eac t ion i n seawater by formation of chelated complexes between t h e i r C ^ - , Cy-hydroxy l -2+ 2 + groups and the a l k a l i n e - e a r t h d iva lent cat ions (Ca , Mg ) of seawater. Such complexation enhanced t h e i r p h o t o l y t i c degradation rates to l eve ls achieved by these p ter id ines at pH 10 i n the absence of seawater s a l t s . The photo lys i s of the 6-hydroxylated p ter id ines (xanthopterin, oxylumazine) i n seawater showed evidence of intermediate formation of the corresponding 6,7-dihydroxylated d e r i v a t i v e s . It was concluded that the p ter id ines and u r i c a c i d may undergo considerable chemical turnover , without b i o l o g i c a l i n t e r v e n t i o n , i n the marine environment, whi l s t the more r e f r a c t o r y purines would require b i o l o g i c a l agencies for s i g n i f i c a n t breakdown and r e u t i l i z a t i o n . v i . TABLE OF CONTENTS. PAGE ABSTRACT i i i TABLE OF CONTENTS v i LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENTS x v i i i INTRODUCTION 1 MATERIALS AND METHODS 8 The Compounds 8 Paper Chromatography 8 Preparation and Observation of Solutions 10 F i r s t Order Reactions 13 Section I. MATERIALS AND METHODS 'DARK REACTION' 13 General Studies 13 S p e c i f i c Studies 14 Oxylumazine dark oxidation 14 1) Seawater Media 14 .2) Sodium Chloride Media 16 3) Trace Metal Effects 16 Section I I . MATERIALS AND METHODS 'PHOTOLYSIS' 18 General Photolysis Information 18 The Light Source 18 RESULTS 20 Pteridines 20 Dark Reactions 20 Chelation 25 v i i . PAGE Photolysis 27 The Light Source 27 Light Reactions - 'Photolysis' 28 Purines 33 Dark Reactions 33 Light Reactions - 'Photolysis' 34 DISCUSSION 36 Theoretical Considerations 36 Pteridines 36 Dark Reactions 37 Chelation 50 Light Reactions 52 Purines 59 Significance of the Light Source Used 61 B i o l o g i c a l and Ecological Significance of the Results 62 Pteridines 62 Purines 63 TABLES 66 FIGURES 85 LITERATURE CITED 135 APPENDIX 145 A - DEFINITIONS 145 B - EXPERIMENTAL MEDIA 147 C - ROUTINE STERILITY CHECKS 150 v i i i . LIST OF TABLES. TABLE Page I S o l u b i l i t y of pteridines and purines i n water at 20°C. 66 I I Summary of molar e x t i n c t i o n c o e f f i c i e n t s (£x IQ~ ) of pteridines at t h e i r c h a r a c t e r i s t i c absorption maxima (X . ) i n the 295-400nm range. 67 I I I Summary of molar e x t i n c t i o n c o e f f i c i e n t s ( £ x 10 ) of purines at t h e i r c h a r a c t e r i s t i c absorption maxima (X.) i n the 225-300nm range. 69 IV Dark reaction rates of pteridines i n various media at room temperature. 71 V The values of oxylumazine dark oxidation products and authentic compounds on paper chroma-tograms. 72 VI F i r s t order reaction rates and reaction stoichemetry for the oxylumazine dark oxidation i n various media. 73 VII Fe^ + and Cu^ + analysis of f i l t e r e d media used for oxylumazine dark oxidation studies. 74 2 + 2 + V I I I The eff e c t of Fe and Cu on the oxylumazine dark oxidation reaction i n various media. 75 IX K i n e t i c evidence for the formation of leucopterin from the dark reaction of xanthopterin i n various media. 76 X Pho t o l y t i c decomposition rates of dioxylumazine i n various media at room temperature under cool-white fluorescent lamps. 77 IX. TABLE Page XI Photolytic decomposition rates of is©xanthopterin i n various media at room temperature under cool-white fluorescent lamps. 78 XII P h o t o l y t i c decomposition rates of leucopterin i n various media at room temperature under cool-white fluorescent lamps. 79 XIII Ph o t o l y t i c decomposition rates of lumazine and p t e r i n i n various media at room temperature under cool-white fluorescent lamps. 80 XIV P h o t o l y t i c decomposition rates of oxylumazine i n various media at room temperature under cool-white fluorescent lamps. 81 XV Pho t o l y t i c decomposition rates of xanthopterin i n various media at room temperature under c o o l -white fluorescent lamps. 82 XVI Dark rea c t i o n rates of purines i n NSM-medium at room temperature. 83 XVII Photolytic decomposition rates of purines i n NSM-medium at room temperature under cool-white fluorescent lamps. 84 x. LIST OF FIGURES. FIGURE PAGE 1 Dark oxidation of oxylumazine i n synthetic seawater. 85 2 Changes i n the absorption spectrum of oxylumazine i n synthetic seawater with the indicated periods of storage i n darkness at room temperature under, aseptic conditions. 86 3 The e f f e c t of sodium chloride concentration i n 0.05M sodium phosphate buff e r (pH 7.8-7.9) on the dark oxidation rate of oxylumazine. 87 4 Changes i n the absorption spectrum of oxylumazine i n 1.0M sodium chloride (buffered at 7.9) with the indicated periods of storage i n darkness at room temperature under aseptic conditions. 88 5 Absorption spectra of dioxylumazine and oxyluma-zine i n pH 8.0 phosphate buffer and a f t e r a c i d i -f i c a t i o n to pH 1.0. 89 6 Dark oxidation of oxylumazine i n 1.0M NaCl s o l u t i o n buffered at pH 7.9. 90 7 Changes i n the major absorption peaks of dioxy-lumazine, leucopterin, and 6,7-dihydroxypteridine i n various media by divalent metal ions. 91 FIGURE 10 11 12 13 14 Absorption spectra of 6 ,7 -d ihydroxypter id ine i n pH 8.0 phosphate buf fer , i n PAPA seawater and a f t e r a c i d i f i c a t i o n to pH 1.0. Emission spectrum of cool-white f luorescent lamps used for the photolys is experiments. Absorption spectrum of "Kimax" brand glass from the photolys is f l a s k s . The e f fec t of pH on the p h o t o l y t i c r eac t ion rate of dioxylumazine. The effect of pH on the p h o t o l y t i c reac t ion rate of l eucopter in . The effect of pH on the p h o t o l y t i c reac t ion rate of oxylumazine. The effect of pH on the p h o t o l y t i c reaction rate of xanthoDterin. PAGE 92 93 94 95 96 97 98 15 16 The effect of pH on the p h o t o l y t i c r eac t ion rate of i soxanthopter in . 99 Changes i n the absorpt ion spectrum of dioxy-lumazine i n PAPA seawater with the ind ica ted per iod of i l l u m i n a t i o n by cool-white f luorescent lamps under asept ic condi t ions . 100 x i i . FIGURE PAGE 17 Changes i n the absorption spectrum of i s o -xanthopterin i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. 101 13 Changes i n the absorption spectrum of lencopterin i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. 102 19 Changes i n the absorption spectrum of oxylumazine i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. 103 20 Changes i n the absorption spectrum of xanthopterin i n PAPA, seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. 21 Changes i n the absorption spectrum of lumazine i n FAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. Also, the absorption spectrum of lumazine at zero hours aft e r a c i d i -f i c a t i o n to pU 1.0. 105 x i n . F I G U R E PAGE 22 Changes i n the absorpt ion spectrum of p t e r i n i n PAPA seawater with the ind ica ted periods of i l l u m i n a t i o n by cool-white f luorescent lamps under asept i c cond i t i ons . A l s o the absorpt ion spectrum of p t e r i n at zero hours a f t e r a c i d i f i -ca t ion to pH 1 . 0 . 106 23 Changes i n the absorpt ion spectrum of oxy-lumazine i n pH 7 .0 sodium phosphate buf f er with the ind ica ted periods of i l l u m i n a t i o n by c o o l -white f luorescent lamps under asept ic conditons. 107 24 Concentration versus time for the p h o t o l y t i c decomposition of oxylumazine and for the appearance of dioxylumazine i n FAPA synthet i c and modified seawater under asept ic cond i t i ons . 108 2 5 Concentrat ion versus time for the p h o t o l y t i c decomposition of oxylumazine and f o r the appearance of dioxylumazine i n pH 10.0 (sodium carbonate) and pH 8 . 0 (sodium phosphate) buffers under asept i c c o n d i t i o n s . 109 26 Concentration versus time for the p h o t o l y t i c decomposition of oxylumazine and for the appearance of dioxylumaze i n pH 7 .0 (sodium phosphate) and pH 4 . 0 (sodium acetate) buf fer under a sep t i c cond i t ions . 110 x i v . FIGURE PAGE 27 Concentration versus time f o r the pho t o l y t i c decomposition of oxylumazine and f o r the appearance of dioxylumazine i n pH 5.4 (sodium acetate) buffer under aseptic conditions. 111 28 Changes i n the absorption spectrum of xanthopterin i n pH 7.0 sodium phosphate buffer with the indicated periods of il l u m i n a -t i o n by cool-white fluorescent lamps under aseptic conditions. 112 29 Absorption spectra of leucopterin and xanthopterin i n pH 8.0 phosphate buffer and a f t e r a c i d i f i c a t i o n to pH 1.0. 113 30 Concentration versus time f o r the ph o t o l y t i c decomposition of xanthopterin and f o r the appearance of leucopterin i n PAPA and synthetic seawater under aseptic conditions. 114 31 Concentration versus time f o r the p h o t o l y t i c decomposition of xanthopterin and f o r the appearance of leucopterin i n modified seawater and ion mix medium under aseptic conditions. 115 32 Concentration versus time f o r the pho t o l y t i c decomposition of xanthopterin and f o r the appearance of leucopterin i n pH 10.0 (sodium carbonate) and pH 8.0 (sodium phosphate) buffers under aseptic conditions. 116 Concentration versus time f o r the p h o t o l y t i c decomposition of xanthopterin and f o r the appearance of leucopterin i n pH 7.0 (sodium phosphate) and pH 4.0 (sodium acetate) buffers under aseptic conditions. Concentration versus time f o r the decomposi-t i o n of xanthopterin and f o r the appearance of leucopterin i n pH 5.0 (sodium acetate) buffer under aseptic conditions. Absorption spectrum of ispxanthopterin i n pH 8.0 sodium phosphate b u f f e r and a f t e r a c i d i f i c a t i o n to pH 1.0. Temporal changes i n the absorption of adenine, guanine, hypoxanthine, and xanthine i n NSM-medium under aseptic conditions while under il l u m i n a t i o n or i n dark storage at room temperature. Changes i n the absorption spectrum of adenine and guanine under aseptic conditions i n NSM-medium with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps or storage i n darkness at room temperature. Changes i n the absorpt ion spectrum of hypoxanthine and xanthine under asept i c condit ions i n NSM-medium with the ind ica ted periods of i l l u m i n a t i o n by cool-white f luorescent lamps or storage i n darkness at room tempterature. Concentration versus time for the decomposition of u r i c a c i d under asept i c condit ions i n NSM-medium during i l l u m i n a t i o n by cool-white f luorescent lamps or storage i n darkness at room temperature. Changes i n the absorpt ion spectrum of u r i c a c i d under asept i c condit ions i n NSM-medium with the ind ica ted periods of i l l u m i n a t i o n by cool-white f luorescent lamps or storage i n darkness at room temperature. Diagram of the xanthopterin molecular species and percentage hydrated expected at various pH va lues . Diagram of the oxylumazine molecular species and percentage hydrated expected at various pH values . Proposed dark reac t ion scheme for the s t o i c h i o m e t r i c conversion of oxylumazine to dioxylumazine and xanthopter in to l e u c o p t e r i n . Diagram of the dioxylumazine molecular species expected at various pH va lues . Diagram of the l eucopter in molecular species expected at various pH va lues . Diagram of the 6,7-dihydroxypteridine molecular species expected at various pI-T va lues . Proposed and known five-membered r i n g chelates between p ter id ine der iva t ives and d iva lent metal ca t ions . Diagram of the i soxanthopter in molecular species expected at various p!-I va lues . Diagram of the molecular species of lumazine and p t e r i n expected at various pH va lues . Absorpt ion spectra of 6-hydroxy-2,4,5-t r iaminopyr imid ine , cy tos ine , pyrazinamide, and 2 - p y r a z i n o l i n PAPA seawater. x v i i i . ACKNOWLEDGEMEHTS. I wish to express my s incere apprec ia t ion to Dr . N . J . A n t i a , F i s h e r i e s Research Board of Canada, Vancouver Laboratory , under whose superv i s ion th i s study was conducted, and for h i s va luable advice and c r i t i c i s m during the research and preparat ion of t h i s manuscript . To Professor A. A l b e r t , John Curt in School of Medica l Research, A u s t r a l i a n Nat ional U n i v e r s i t y , Canberra, for his kindness i n prov id ing samples of c e r t a i n synthet i c p ter id ines and for o f f e r i n g invaluable advice and information on p t e r i d i n e chemistry whenever i t was needed. To Mr. J.Y. Cheng and the other s t a f f members of the F i s h e r i e s Research Board of Canada, Vancouver Laboratory for t h e i r advice and ass is tance with the various aspects of t h i s s tudy. To the D i r e c t o r of the F i sher i e s Research Board of Canada, Vancouver Laboratory for author iz ing the laboratory space and use of i t s f a c i l i t i e s which made t h i s study p o s s i b l e . To my Mother, Mrs . F . G . Landymore and to Mrs . J . N . C . Whyte for the many hours spent typing t h i s manuscript . To Dr . P . A . L a r k i n for accept ing me as a graduate student on behal f of the Department of Zoology and for organiz ing the cooperation with the F i sher i e s Research Board of Canada, which made t h i s i n v e s t i g a t i o n p o s s i b l e . To the F i s h e r i e s Research Board of Canada for prov id ing f i n a n c i a l ass i s tance during th i s study. F i n a l l y , but f i r s t l y , to my wife Marlene for l i s t e n i n g to x i x . the many problems I had during the preparation of t h i s manuscript. 1. INTRODUCTION. Of the many subjects covered by marine chemistry, that of dissolved organic matter i n seawater appears to be the least understood (Duursma, 1965). The dissolved portion of the organic matter i n seawater i s defined as that which passes a type HA M i l l i p o r e R f i l t e r with a 0.45-micron pore s i z e (Hood, 1963; Ogura, 1970). Analyses of the t o t a l organic carbon i n s o l u t i o n i n the oceans have been made by a number of investigators, and values i n the range 0.4-6.0 mg C/L have been obtained ( J e f f r e y and Hood, 1958; Hood, 1963). On the other hand, the t o t a l dissolved organic nitrogen and organic phosphorous have been estimated at 0.03-0.40 mg N/L and 0-9.0 pg P/L r e s p e c t i v e l y (Hood, 1963). Concentrations of the t o t a l dissolved material have not r e a l l y been determined, because i t s chemical structure i s unknown and the factors for conversion of carbon, nitrogen, and phosphorous to t o t a l organic matter canot be adequately applied. It has been estimated by Steemann (1955) and Duursma (1961) that dissolved organic carbon exceeds the concentration of l i v i n g and p a r t i c u l a t e organic carbon by a factor of eight or more. Plunkett and Rakestraw (1955) and Holm-Hansen et a l . (1966) reported that the dissolved organic carbon i s not d i s t r i b u t e d uniformly e i t h e r v e r t i c a l l y or h o r i z o n t a l l y i n the oceans. However, i f a figu r e of 1 mg C/L i s taken as a very rough measure of-the average concentration of dissolved carbon throughout the oceans, the t o t a l mass of dissolved organic carbon may be estimated at 12 ca. 1.37 x 10 metric tons. This i s a f a c t o r of ten greater than the p a r t i c u l a t e d e t r i t a l carbon (Parsons, 1963) and the comparable annual production of phytoplankton of 1.5 x 10*"*" 2. metric tons (Riley, 1953). This i s also one hundred times greater than the production of land plants, ca_. 1.5 x 10*^ metric tons (Fox, i960). Thus the largest quantity of organic carbon upon t h i s planet, outside of that i n sediments and s o i l (estimated at 3.8 x l O 1 ^ metric tons; Hunt, 1962) i s dispersed i n the ocean. Of the several obstacles t o the study of the molecular nature of the dissolved organic matter i n seawater, the f i r s t and greatest i s the problem of e f f i c i e n t l y extracting quantities of 6 mg C/L or less from an aqueous so l u t i o n containing ten thousand times as much inorganic s a l t s . To obtain s u f f i c i e n t material f or chemical ana l y s i s , l i t e r quantities of seawater must be processed during which destruction of l a b i l e compounds of b i o l o g i c a l o r i g i n by heat or other treatments i s d i f f i c u l t to prevent. Further i s o l a t i o n problems (such as b a c t e r i a l decomposition, adsorption on container surfaces, etc.) have made i t v i r t u a l l y impossible to monitor s u f f i c i e n t seawater samples to reach any s a t i s f a c t o r y conclusions concerning the chemical nature of the organic compounds present i n the oceans. Many methods have been t r i e d to i d e n t i f y the chemical nature of these compounds. J e f f r e y and Hood (1958) have evaluated the various methods used and concluded that co-p r e c i p i t a t i o n with f e r r i c hydroxide i s the most promising method, but removal of f e r r i c hydroxide s t i l l presents a problem. A number of reviews on the probable i d e n t i t y of the dissolved organic matter have been published by Vallentyne (1957), Hood (1963), Provasoli (1963), Duursma (1961, 1965) and Pocklington (1971). Amino acids, peptides, 3. proteins, carbohydrates, l i p i d s , organic acids and vitamins' have a l l been reported. These compounds exis t i n variable amounts, with the highest concentrations found i n oceanic surface and off shore waters and generally accompanying the highest b i o l o g i c a l production found (Parsons and S t r i c k l a n d , 1962); however, accurate determination of concentrations and characterization has been very l i m i t e d . The b i o l o g i c a l s i g n i f i c a n c e of these compounds i s not yet understood, but Baylor et a l . (1963) have suggested that they may constitute a 'reservoir* of food for marine organisms. The vitamins present i n trace amounts, have been most d i l i g e n t l y sought for by use of bioassay techniques (Belser, 1963), with vitamin B ^ occupying the attention of most investigators due to i t s requirement f o r growth by many planktonic organisms (Droop, 1957). Work on other vitamins has been very l i m i t e d , and the occurrence of thiamine, n i a c i n , and b i o t i n have been occasionally demonstrated. Many other s p e c i f i c types of compounds have been documented, for example, toxins from 'red t i d e ' organisms (Abbot and Ballentyne, 1957), plant growth hormone-like substances (Bentley, 1960), humic acids (Armstrong et a l . , 1961) phenolic compounds (Craigie et aJL., 1964; Degenes et a l . , 1964), and pyrimidines and purines (Belser, 1963). The following possible origins of dissolved organic compounds may be l i s t e d : ( l ) drainage from land i n t o coastal marine areas; (2) secretions or excretions of l i v i n g organisms i n the course of t h e i r metabolism; (3) decomposition of senescent or dead marine organisms; and (4) modifications of such decomposed matter by microorganisms and min e r a l i z a t i o n processes. In the seas, the phytoplanktonic organisms constitute the bulk of l i v i n g organic matter, with less than 10% converted i n t o animal tiss u e (Duursma, 1965). Consequently, t h e i r excretion and decomposition may be expected to make the largest contribu-t i o n to the pool of dissolved organic matter i n seawater. Phytoplankter c u l t u r e studies have given evidence of production of diverse organic compounds i n seawater media (Duursma, 1961; Hellebust, 1965), and such compounds may generally be expected to occur i n marine areas containing these organisms. Other types of organic compounds w i l l be produced by senescent or decomposing phytoplankton, which w i l l be c o n t i n u a l l y undergoing modification by b a c t e r i a l action or by mineralization processes. Resistant compounds become concentrated i n the sediment (Vallentyne, 1957) where further modifications appear to be f a c i l i t a t e d from surface a c t i o n . The contribution of organic compounds from the sediments i s not known and i s presumed to be of minor importance. Run-off from land drainage and from sewage i s known t o contain many organic compounds (Valientyne, 1957), however few of these compounds have ever been found i n the marine environment. The r o l e s of bact e r i a , yeast, and fungi i n the metabolic turnover of dissolved organic matter i n seawater are now well documented (Wood, 1958, 1965). An important function of t h i s r o l e appears to be the regeneration of inorganic nutrients and organic growth factors (vitamins; Burkholder, 1959) required f o r growth of phytoplankton. However, phytoplanktors themselves have also been implicated i n the metabolism of dissolved organic matter. A f a i r number of phytoplanktors have recently been shown to l i v e heterotrophically, that i s , using organic compounds for n u t r i t i o n . This heterotrophy may be f a c u l t a t i v e , obligate, 5. or combined with photoautotrophy (see Appendix A). Carbohydrates, organic acids, Krebs-cycle intermediates, amino acids and f a t t y acids have been shown to support t h e i r growth (Danforth, 1962; Cheng and Antia, 1970). The e c o l o g i c a l importance of heterotrophy i s d i f f i c u l t to assess because most studies were eff e c t e d under laboratory conditions, but healthy phytoplankton have been frequently reported below the photic zone, implicating t h e i r s u r v i v a l by heterotrophy, although t h i s may not be n e c e s s a r i l y the case (Antia and Cheng, 1970). Organic nitrogen compounds are often metabolized photo-t r o p i c a l l y by phytoplankton to s a t i s f y t h e i r nitrogen require-ment (Provasoli, 1963; Syrett, 1962; Antia and Chomey, 1968). Compounds such as amino acids, amino sugars, amides, purines, pyrimidines, urea, and some of t h e i r derivatives have served as a nitrogen source f o r growth. The e f f i c i e n t u t i l i z a t i o n of purines by a marine cryptomonad was recently demonstrated (Antia and Chomey, 1968) and the suggestion was made that such a process may have e c o l o g i c a l s i g n i f i c a n c e i n the turn-over of purines emanating from excreta and sewage p o l l u t i o n . In t h i s context, i t was of i n t e r e s t to investigate the p o s s i b i l i t y of u t i l i z a t i o n by phytoplankters of the s t r u c t u r a l l y r e l a t e d pteridines (see Appendix A) also known to occur i n many forms of aquatic l i f e (see below). However, such preliminary studies on pteridines revealed an unexpected aspect of t h e i r possible turn-over i n the marine environment. Several of the pteridines investigated gave evidence of chemical degradation or modification i n seawater, mostly under the influence of l i g h t , without the a c t i o n of any b i o l o g i c a l agency. The f i r s t observation was made 6. with a s o l u t i o n of xanthopterin i n seawater, which showed a v i s i b l e change i n color without any phytoplankton growth, and t h i s change i n light-absorption properties was traced to the chemical action of seawater combined with l i g h t on the pteridine maintained under s t e r i l e conditions. The s t r u c t u r a l s i m i l a r i t y of purines to pteridines suggested that they might behave s i m i l a r l y i n seawater and that previous observations of t h e i r u t i l i z a t i o n by phytoplankters might be misleading i n that the organisms may be a c t u a l l y using seawater-degraded or modified products rather than the in t a c t purines. Accordingly, i t was considered desirable to make a systematic i n v e s t i g a t i o n of the effects of the s a l t s of seawater with and without l i g h t on the chemical s t a b i l i t y of several pteridines and purines. Possible origins of pteridines and purines are p l a n t s , animals, and microorganisms. Blakely (1969), i n his monograph, presents sources of pteridines as insect wings and eyes, amphibian s k i n and eyes, mammalian blood and urine, r e p t i l e s k i n , plants including algae, f i s h s k i n , various crustaceans, bacteria and protozoa. In f i s h and amphibia, pteridines have been implicated i n melanin synthesis (Blakely, 1969) while i n plants and algae a 6-substituted pteridine has been i s o l a t e d and i s thought to be involved i n the photosynthetic apparatus ( F u l l e r et a l . , 1969). In the blue-green alga Anacystis nidulans, cold shock releases b i o p t e r i n (6-dihydroxypropylpterin) into the medium (Forrest et a l . , 1957), t h i s pteridine has also been found i n other algae ( H a t f i e l d et a l . , 1961). On the other hand, purines which are also p l e n t i f u l , are found i n water from land drainage (Vallentyne, 1957), i n f i s h s k i n ( F u j i i , 1969; Lee et a l . , 1969), and i n plant and animal excretion products (Bendich, 1955). The presence of purines i n seawater has been shown by Belser (1963), and L i t c h f i e l d et al., (1966), however pt e r i d i n e s , u n l i k e purines have not been looked for i n seawater u n t i l very re c e n t l y (Albert, 1970). MATERIALS AND METHODS 8. General Mater ia l s and Methods  The Compounds The p ter id ines and purines used i n th i s study were reagent grade compounds or authent ic specimens obtained from the fo l lowing sources and were used without fur ther p u r i f i c a t i o n . The p ter id ines were: 2,4 -dihydroxypteridine ( lumazine) , 2-amino-4-hydroxypteridine ( p t e r i n ) , and 2-amino-4, 7 -d ihydroxypter id ine ( i soxanthopterin) from A l d r i c h Chemical C o . , Milwaukee, Wisconsin; 2-amino-4, 6 -d ihydroxypter idine (xanthopterin) from Mann Research Laboratories Inc . , New York; 2-amino-4,6,7-trihydroxypteridine ( l eucopter in) from Calbiochem, Los Angeles, C a l i f o r n i a ; 6,7 -dihydroxypteridine, 2,4,6 - tr ihydroxypteridine (oxylumazine) and 2,4,6,7-tetrahydroxy-p t e r i d i n e (dioxylumazine) from Prof . A . A l b e r t , John C u r t i n School of Medical Research, A u s t r a l i a n Nat ional U n i v e r s i t y , Canberra. The purines were: 6-aminopurine (adenine), 6-hydroxypurine (hypoxantnine), 2-amino-6-hydroxypurine (guanine), and 2,6,8-tr ihydroxypurine ( u r i c ac id ) from Mann Research Laborator i e s , New York; and 2,6-dihydroxypurine (xanthine) from Sigma Chemical C o . , S t . Lou i s , M i s s o u r i . Paper Chromatography Whatman No. 3MM chromatographic paper was used i n a l l chroma-tographic analyses . A l l chromatograms were developed by descending solvent flow at room temperature (22-25°C), protected from d irec t l i g h t . The fo l lowing solvent systems were used for developing chromatograms as required: (I) the organic upper layer of butan-l - o l : a c e t ic a c i d : water (4:1:5, v/v); (II) propan-2-ol: concen-t r a t e d HC1: water (4:1:5, v/v); (III) propan-l-ol: 0.5N NH^OH (2:1, v/v); (IV) butan-l-ol: a c e t i c a c i d : water (12:5:3, v/v); (V) propan-l-ol: water:15N NH^OH (40:20:1, v/v); and (VI) aqueous 3% (0.56M) NH^Cl so l u t i o n . The developed chromatograms were a i r -dried at room temperature i n the dark and scanned under UV-ra d i a t i o n at 366 and 253.7 nm for UV-fluorescing and -absorbing spots. The detection of UV-fluorescing spots was enhanced by exposure of the chromatograms to ammonium hydroxide fumes. The homogeneity of a l l pteridines and purines studied was established by paper chromatography i n the following solvent systems ( I ) , ( I I ) , and ( I I I ) , shown above. The formation of dioxylumazine from oxylumazine and of leucopterin from xanthopterin i n the various reaction media was confirmed by paper chromatographic analysis of the e n t i r e reaction products without deionization and comparing d i r e c t l y with standards dissolved i n the corresponding media (as the values were affected by the various media). Xanthopterin-leucopterin solutions were developed i n the solvent system (I) shown above, whereas the oxylumazine-dioxylumazine solutions were developed i n the following solvent systems (IV), (V), and (VI) , shown above, known to give good separations of oxylumazine from dioxylumazine (Levy and McNutt, 1962). Where required the dioxylumazine and leucopterin bands were eluted from preparative chromatograms with 3% NH^OH for sp e c t r a l comparison with authentic samples. 10. Preparation and observation of Solutions. The compositions of the media used i n the study of the pteridines and purines are l i s t e d i n Appendix B. For the p t e r i d i n e studies, PAPA seawater, synthetic seawater, modified seawater, aqueous buffers and metal ion mixes were used. However, the purines were studied using only one medium (NSM-medium). This medium had previously been used f o r nitrogen u t i l i z a t i o n studies of Hemiselmis  virescens (Antia and Chorney, 1968) which had involved the use of purines as a nitrogen source. The following buffers of strength 0.05M were used f o r the pteridine studies and were: sodium acetate buffer pH 4.0,5.0,5.4 sodium phosphate buffer pH 6.0,7.0,8.0 Tris * - H C l buffer pH 7.0,8.3 bor i c acid-sodium borate buffer pH 9.0 sodium carbonate buffer pH 10.0 A l l buffers were prepared using g l a s s - d i s t i l l e d water following the methods given by Gomori (1955). *Sigma Chemical Co., St. Louis, Missouri. The other chemicals used were 'Baker Analyzed' Reagents, J.T. Baker Chemical Co., P h i l l i p s b u r g , N . J . The concentration of pteridines and purines i n s o l u t i o n was generally at the l e v e l of 0.04mM; i n some instances lower concentra-tions were used because of s o l u b i l i t y problems. In Table I, the concentrations used are given for each compound along with the maximum permissible concentration f o r each compound to remain i n s o l u t i o n at 20°C i n d i s t i l l e d water. Solutions of these compounds 11. were prepared, using the media i n Appendix B or the bu f f e r s , being studied, by warming u n t i l v i r t u a l l y complete d i s s o l u t i o n was obtained. The solutions were cooled to room temperature and i f a volume change had occurred from evaporation during heating, g l a s s - d i s t i l l e d water was added to correct the l o s s . The solutions were then f i l t e r - s t e r i l i z e d (0.2-micron pore-size Gelman M a t r i c e l f i l t e r i n s t e r i l e disposable p l a s t i c units from Nalge Co., Rochester, N.Y.). Aliquots (50 ml.) of these solutions were maintained a s e p t i c a l l y i n 125-ml capacity screwcapped Erlenmeyer f l a s k s . These fla s k s had previously been s t e r i l i z e d by auto-claving 20 min. at 120°C and 15 p s i . For the photolysis studies, three Erlenmeyer flasks were used per experiment. The f i r s t f l a s k ('solvent control') contained the s t e r i l i z e d medium i n which the p t e r i d i n e or purine was to be studied. This 'solvent' was used i n the reference c e l l when absorption spectra were determined. The other f l a s k s , the 'dark reaction c o n t r o l ' and 'photolysis t e s t ' each contained 50-ml aliquots of the 100-ml s o l u t i o n prepared for study. For the dark reaction studies, only a 50-ml s o l u t i o n was prepared and one f l a s k - equivalent to the above 'dark reaction control* - was used alongside a 'solvent c o n t r o l ' . Solutions of 0.04mM were chosen as the best concentration l e v e l of the pteridines and purines f o r ease and accuracy of measurement. At t h i s concentration l e v e l , the absorption spectrum (200-500nm) of any solu t i o n studied was determined d i r e c t l y i n a 1-ml capacity, 1-cm l i g h t path s i l i c a cuvette and the r e l a t i v e error of the i n i t i a l absorabnce readings at the s p e c i f i c wave-length used to monitor each experiment approached i t s smallest value (Ewing, 1969). Reactions of the pteridines or purines i n these solutions were followed by p e r i o d i c observation of changes i n t h e i r absorption spectra (200-500nm f o r pteridines and 200-400nm f o r purines), which were determined on 1-ml aliquots at the o r i g i n a l pH of the solutions and then at pH ca. 1.0 obtained by incorporating one drop of concentrated HC1 int o the sample i n the cuvette. The frequency of observations was determined by the rate of absorbance change of the t e s t s o l u t i o n r e l a t i v e to i t s con t r o l . A l l absorption spectra were recorded on a Beckman DB-G grating spectrophotometer employing a 10" Beckman recorder. Precise values of the absorbance at s p e c i f i c wave-lengths were read on a Beckman DU spectrophotometer. The wave-lengths chosen for k i n e t i c measurements were those of the c h a r a c t e r i s t i c absorption maximum nearest 400nm f o r the pteridines and 300 nm for the purines. Wherever doubtful,sudden decreases i n sp e c t r a l absorption were checked for possible delayed p r e c i p i t a t i o n . If p r e c i p i t a t i o n was noticed,a new t e s t was s t a r t e d with a lower concentration of pteridine or purine. If no p r e c i p i t a t e was v i s i b l e , the s o l u t i o n was heated to see i f the absorbance increased from s o l u b i l i z a t i o n of a possible fine p a r t i c u l a t e dispersion; only when t h i s did not occur, then the reaction was considered to be t r u l y taking place. In the preparation of p t e r i d i n e and purine solutions, i t was not practicable to r o u t i n e l y weigh an exact amount of the compounds The volume of solutions prepared were 50 or 100 ml, thus requiring between 0.2-0.4 or 0.5-0.7 mg of compound, re s p e c t i v e l y . Therefore molar e x t i n c t i o n c o e f f i c i e n t s were used to determine the exact concentrations prepared i n each case. These c o e f f i c i e n t s were determined experimentally and were compared with l i t e r a t u r e values (see Table II and Table III) f o r the absorption spectra at pH ca. 1.0. The sample aliquots required for spectrometric observation were withdrawn a s e p t i c a l l y from the t e s t flasks with dry-heat s t e r i l i z e d 9-inch, disposable c a p i l l a r y Pasteur pipets. Routine s t e r i l i t y checks of the reaction solutions for microbial contami-nation were made by inoculating one to two drops in t o 2.5 ml aliquots of STP and ST 3 media of Tatewaki and Provasoli (1964), (see Appendix C), and incubating i n darkness fo r 3 weeks at room temperature. F i r s t Order Reactions In the tabulated r e s u l t s , f i r s t - o r d e r reaction rates are also expressed i n terms of zero-order reaction rates f o r d i r e c t comparison with those pteridines showing zero-order reaction rates only. The zero-order reaction rates were calculated from the corresponding f i r s t - o r d e r reaction k i n e t i c s at a concentration of 0.04mM (the slope of a tangent to the reaction rate curve at the i n i t i a l pteridine concentration) and these zero-order values are only v a l i d at t h i s concentration. Section I. MATERIALS AND METHODS 'DARK REACTION' General Studies The 'dark reaction c o n t r o l ' f l a s k used i n the photolysis studies served to determine the e f f e c t s of the media on the pteridines and purines without the influence of l i g h t . This r e a c t i o n i f any, was l a b e l l e d 'dark reaction'. The preparation and observation of these series of flasks has been described under the General Methods section. Care was taken to exclude l i g h t by wrapping these flasks with aluminum f o i l and incubating them i n a dark cupboard at room temperature (22-25°C) for the duration of the experiment. The 'solvent c o n t r o l ' flasks were treated as described under Section II of Materials and Methods excepting the case of s p e c i f i c studies described below. - S p e c i f i c Studies  Oxylumazine dark oxidation The decrease i n absorbance values of the oxylumazine 'dark reaction controls* i n synthetic seawater media, prompted an i n v e s t i g a t i o n into the nature of the dark reaction. This s p e c i f i c study w i l l be considered under three categories, based on the media involved i n the experiment: ( l ) seawater media, (2) sodium chloride media, and (3) trace-metal ion e f f e c t s from both these types of media. In part 1, 100-ml solutions were prepared i n each medium; 50-ml f o r photolysis and 50-ml for dark reaction studies. However, i n parts 2 and 3, only 50-ml solutions were prepared. In these two parts (2 and 3), the 'solvent c o n t r o l ' f l a s k , although not wrapped with aluminum f o i l , was stored with the 'dark reaction' f l a s k i n the cupboard. 1) Seawater Media The dark oxidation was followed i n synthetic, modified, and PAPA seawater. The changes i n absorption values of each t e s t s o l u t i o n at the medium pH and pH ca. 1.0 (obtained a f t e r a c i d i f i c a ' t i o n ) were used to calculate the concentrations of oxylumazine and and the reaction product, dioxylumazine. The following equations, based on Beer's Law and additive absorbances, were used to calculate the concentrations: A m = [DIOLU] JL + [OLU] £° <L A a = [DIOLU] + [OLU] £° * Where A^ = absorbance at medium pH; A^ = absorbance at pH ca. 1.0: m a r — 7 £ = determined molar e x t i n c t i o n c o e f f i c i e n t at medium pH (m) or acid pH (a) for dioxylumazine (d) or oxylumazine (o); [DIOLU] molar concentration of dioxylumazine; [OLU] = molar concentration of oxylumazine; and jL = length i n centimeters of l i g h t path. The concentration curves,in F i g . 1, for oxylumazine and dioxylumazine i n synthetic seawater were constructed using these equations. For example, at 900 hours, A m = 0.046 at 380 nm; A a = 0.080 at 366 nm;£values are taken from Table I I and JL. = 1.0 cm. Substitution of these values i n t o the equations gives: 2 3 0.046 = [DIOLU] x 6,56 x 10 x l . 0 + [OLU] x 6.75 x 10 x 1.0 3 3 0.080 = [DIOLU] x 3.02 x 10 x 1.0 + [OLU] x 5.17 x 10 x 1.0 Solving f o r [OLU] and [DIOLU] , the calculated concentrations found for each compound at 900 hours were: [OLU] = O.OllOmM and [DIOLU] = 0.0l68mM. Si m i l a r calculations were made f o r the other media used. From these concentration curves, the rates of disappearance of oxylumazine and appearance of dioxylumazine were calculated f o r each medium. Note: Calculation of the concentrations of dioxylumazine and oxylumazine not only i n the 'dark reactions' but also i n the 'photolysis reactions' were undertaken using the method described above. In other cases, the concentrations of leucopterin and xanthopterin i n the 'dark reactions' and the 'photolytic reactions' were calculated using the same method, however i n the equations, xanthopterin was substituted f o r oxylumazine and likewise leucopterin f o r dioxylumazine. The e x t i n c t i o n c o e f f i c i e n t s f o r leucopterin and xanthopterin used i n the calculations are shown i n Table I I . 2) Sodium Chloride Media The dark reaction of oxylumazine was followed i n sodium chloride solutions i n the concentration range of 0.1 to 1.4 Molar. Each so l u t i o n was buffered at pH 7.8-7.9 with 0.05M sodium phosphate buffer ( o r i g i n a l l y a pH 8.0 buffer was prepared, but the i o n i c strength of the s a l t s a l t e r e d the pH to between 7.8 to 7.9). Also, the effects of the various ions i n seawater were studied i n 0.5M sodium chloride solutions buffered at pH 7.9-8.0, using concentrations corresponding to those reported by Lyman and Fleming (1940) for synthetic seawater. The ef f e c t of sodium ions was studied by s u b s t i t u t i n g potas-sium chloride i n place of sodium chloride at one concentration (0.5M) and pH 8.0 obtained with 0.05M potassium phosphate buffer. 'Baker Analyzed' reagent grade s a l t s were used throughout. Reaction rates were calculated as described before, using Rvalues l i s t e d i n Table I I . 3) Trace Metal Effects The importance of trace-metal ions present as normal constituents or as contaminants i n the above media was recognized 17. during these studies from i n h i b i t i o n of the dark reaction by the complexing agent ethylenediaminetetra-acetic a c i d (EDTA) and i t s reversal by subsequent incorporation of trace-metal ion, EDTA (disodium s a l t , Eastman Organic Chemicals) was added to synthetic seawater and 0.5M sodium chloride solutions buffered at pH 8.0 with sodium phosphate buffer. A concentration of 5.0uM EDTA was used to give a 5-fold excess over the estimated concentration of heavy metals. Reversal of the r e s u l t i n g i n h i b i t i o n was attempted 2+ 2 + i n both media by the addition of 5.0uM of Fe or Cu The s a l t s used were F e S O ^ N H ^ S O ^ ^ O and CuSO^«5H 20 ('Baker Analized' 2+ 2+ 2 + reagent grade chemicals). Also the e f f e c t of Fe and Fe -Cu on oxylumazine prepared i n glass d i s t i l l e d water buffered at pH 8.0 with 0.05M sodium phosphate buffer, was also studied. „ r--, ~ n 2+ 2 + The concentrations used, 0.57 and 0.l9uM for Fe and Cu respectively, approximated the concentration of these trace-metals present as contaminants i n a 0.5M sodium chloride s o l u t i o n buffered at pH 8.0 with 0.05M phosphate buffer. 2+ 2 + The concentrations of Fe and Cu i n a l l seawater and NaCl media used were determined by the methods of St r i c k l a n d and Parsons (1964), with the following differences. The spectro-photometry measurements were made with a 1-cm l i g h t path instead of the 10-cm l i g h t path described i n the methods, r e s u l t i n g i n a ten - f o l d reduction of s e n s i t i v i t y . In the copper analysis, flushing the sodium diethyldithiocarbamate s o l u t i o n with nitrogen gas, prevented a i r oxidation and extended the s t a b i l i t y of the sol u t i o n to about three weeks. A l l sample media were membrane-f i l t e r e d (Millipore RHA, 0.45-micron pore size) before a n a l y s i s . 18. The f i l t e r was pre-washed with a small volume of the media to be analyzed, before the media f o r the analysis was f i l t e r e d . Section II. MATERIALS AND METHODS'PHOTOLYSIS' General Photolysis Information Preparation and observation of the pteridine and purine solutions were described under the section on General Materials and Methods. The storage of the 'dark reaction c o n t r o l f l a s k ' was described i n Section I, of materials and methods 'Dark Reaction'. The other two f l a s k s , the 'solvent c o n t r o l ' and the 'photolysis f l a s k ' , were both placed under the l i g h t source i n an incubator maintained at 20-22°C. The liftht Source Both the 'solvent c o n t r o l ' and photolysis flasks were i r r a d i a t e d continuously from above by four cool-white fluorescent lamps (No. F14T12/CW, Sylvania E l e c t r i c Canada, Ltd.), 58.5 cm i n length. The ce n t r a l two l i g h t s , 15 cm apart, and 31 cm above the reaction flasks were p a r a l l e l . The other two l i g h t s , one on each side and p a r a l l e l to the c e n t r a l l i g h t s , were 25.5 cm from them and i n the same plane. This arrangement of l i g h t s was used for a l l the photolysis studies. The spectrum of the cool-white fluorescent lamps was determined by using a fluorescence spectrophotometer (Fluorispec, Model SF-1, Baird-Atomic Inc., Cambridge, Mass.). Only the fluorescence mono-chromator portion of the instrument was u t i l i z e d with the slowest possible scanning speed. The output was recorded by a Moseley s Autograf X-Y recorder (Model 135C, F.L. Moseley Co., Pasadena, C a l i f o r n i a ) . 19. The t o t a l i l l u m i n a t i o n was determined using a Weston Foot-candle l i g h t meter (Model 614, Weston E l e c t r i c a l Instrument Corp., Newark, N.J.). This instrument contained a f i l t e r to correct the photoelectric response so that the meter indicated correct v i s u a l i l l u m i n a t i o n . Also, the t o t a l flux of energy was measured using a YSI-Kettering Radiometer (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio). The. source of l i g h t , i n a l l cases, passed through the glass of the 125-ml screwcapped Erlenmeyer f l a s k s . The absorption of l i g h t (200-750nm) by t h i s glass (Kimax brand) was determined by placing a piece of a f l a s k i n the sample beam of the double beam Beckman DE-G grating spectrophotometer. The e f f e c t of the glass upon the spectra of the fluorescent l i g h t (200-370nm) was also determined. In t h i s case, the glass was placed between the l i g h t source and the f i r s t s l i t of the fluorescence monochromator portion of the spectrophotometer. 20. RESULTS. Pteridines Dark Reactions. The r e s u l t s from the dark reactions of pteridines are presented i n Table IV. From a consideration of t h i s data, various reaction rate patterns were observed. In seawater, the patterns appeared to be related to pter i d i n e structure as follows: (A) Oxylumazine and xanthopterin (both C^-monohydroxylated pteridines) showed the highest reaction rates observed; however the difference between them was s t r i k i n g , the reaction rate f o r oxylumazine being about one order of magnitude greater than that observed f o r xanthopterin. (B) Dioxylumazine and leucopterin (both Cg, C^-dihydroxylated pteridines) showed low reaction rates, while lumazine and p t e r i n (both Cg, Cy-unsubstituted pteridines) and isoxanthopterin (a C^-hydroxylated pteridine) showed no reaction at a l l . In the buffer solutions, no r e l a t i o n s h i p between pteridine structure and reaction rate patterns was observed. In considering the data, i t was noticed that (A) isoxanthopterin and oxylumazine were both unreactive at a l l pH values examined and (B) dioxyluma-zine, leucopterin and xanthopterin a l l showed d i f f e r e n t degrees of a c t i v i t y with no consistent pattern from pH change. In co r r e l a t i n g pH rate patterns with seawater rate patterns, i t appears that, with the exception of oxylumazine, the pteridines tested show seawater rates c l o s e l y p a r a l l e l i n g the pH 8.0-8.3 rates. The case of r e a c t i v i t y of oxylumazine i n seawater as opposed to i t s u n r e a c t i v i t y i n corresponding pH buffers i s exceptional and w i l l be discussed l a t e r . 21. Unl ike the other p t e r i d i n e s , oxylumazine i n seawater was found to undergo a f i r s t - o r d e r dark r e a c t i o n . Corresponding zero-order rates f o r th i s r e a c t i o n were computed and presented i n Table IV to f a c i l i t a t e ready comparison with the zero-order rates of breakdown observed for a l l the other p t e r i d i n e s . A p lo t of the c h a r a c t e r i s t i c absorbance values at pH 7.9 and pH 1.0 for the dark r e a c t i o n of oxylumazine i n synthet ic seawater ind ica ted the occurrence of a complex chemical r e a c t i o n ( F i g . 1) . The progress ive changes i n the absorpt ion spectrum of oxylumazine i n synthet ic seawater ( F i g . 2) not only confirmed the occurrence of a complex chemical r e a c t i o n , but a l s o the appear-ance of a new compound. A f t e r a s u f f i c i e n t l y long period of t ime, the spectrum of dioxylumazine was recognized ( F i g . 2, spectrum at 1647 hours) and, on completion of the r e a c t i o n , the spectra of the product , at two d i f f e r e n t pH va lues , appeared to be i d e n t i c a l with those of authentic dioxylumazine (see F i g . 17, zero-hour spectrum for seawater pH and F i g . 5 f or spectrum at pK 1.0) . In three solvent systems (Table V ) , the seawater reac t ion product and authent ic dioxylumazine showed chromatographic i d e n t i t y , with no other UV-f luoresc ing or UV-absorbing substances being detected. With the i d e n t i f i c a t i o n of dioxylumazine, the concentrations of dioxylumazine and oxylumazine present during progress of the reac t ion were c a l c u l a t e d from the absorbance values obtained and were p l o t t e d versus time ( F i g . l ) . The r e a c t i o n order and rate were determined from t h i s plot f o r syn-t h e t i c seawater, and s i m i l a r plots for the other seawater media. The ef fect of NaCl concentrations at pH 7.3-7.9 on the s t a b i l i t y of oxylumazine was next s tudied and the r e s u l t s ( F i g . 3) 2 2 . showed that (i) 0.25-0.30 M NaCl concentration was required for detectable oxidation, and ( i i ) the reaction rate increased rapidly with NaCl concentration up to 0.6-0.8M and appeared to "level off" at greater salt concentrations. However, the oxidation rate in aqueous NaCl concentration (0.5M) of comparable ionic strnegth to seawater was considerably slower than that found in seawater (Table VI). The progressive changes in the spectrum of oxylumazine in 1.0M NaCl are presented in Figure 4. On completion of the reaction, the spectrum of dioxylumazine was recognized and appeared to be identical with that of authentic dioxylumazine (Fig. 5). In three solvent systems, the NaCl reaction product and authentic dioxylumazine co-chromatographed on paper (see Table V). A plot of the absorbance values at pH 7.9 and pH 1.0 and the concentrations of dioxylumazine and oxylumazine for 1.0M NaCl (Fig. 6) showed a great disparity in the stoichiometry of the conversion of oxylumazine to dioxylumazine between seawater and a l l concentrations of NaCl. Whereas the latter effected a quantitative (1:1) conversion (Fig. 6), the former was repeatedly found to give a (2:1) conversion (Fig. 1). This indicated that in seawater for every mole of oxylumazine oxidized to dioxyluma-zine, another mole of oxylumazine was converted to undetected products. In a comparison of the authentic dioxylumazine (Fig. 17) and the seawater-oxidation product spectra (Fig. 2), the characteristic absorption region was identical; however the overall absorption in the far UV-region (205-240 nm) was significantly greater for the oxidation product than for authentic 23. dioxylumazine. This a d d i t i o n a l absorption i n the f a r UV-region may be a t t r i b u t e d to the u n i d e n t i f i e d products i n the seawater reaction, suggesting that they may not be pteridines (which would be expected to absorb at wavelengths greater than 300nm). The absence of UV-detectable compounds, other than dioxylumazine, on paper chromatograms also supports such an inference. The omission of MgC^, CaC^ and S r C ^ from synthetic seawater gave the same reaction stoichiometry as the whole synthetic seawater and PAPA seawater (Table VI). In an attempt to change the reaction stoichiometry, the various ions at concentrations corresponding to those f o r synthetic seawater were added to 0.5M NaCl. In each case, the stoichiometry remained q u a n t i t a t i v e l y 1:1 f o r each ion or combination of ions studied (Table VII). The rates of the various reactions appeared to have no ef f e c t on the stoichiometry. However, the rates were aff e c t e d by the ions present i n the r e a c t i o n mixture. From the results (Table VI), the anion bromide appeared to be i n h i b i t o r y or po s s i b l y antagonistic to chloride ions i n the reaction, whereas borate appeared to stimulate the reaction. Replacement of sodium by potassium showed no e f f e c t on the reaction rate or stoichiometry. The nature of the e f f e c t of NaCl concentration on the dark oxidation rate of oxylumazine ( F i g . 3) suggested that an impurity i n the s a l t may be involved i n the r e a c t i o n . An analysis of 2 + the media was undertaken for the major two trace-metals, Fe 2 + and Cu , expected to occur i n both seawater and reagent chemicals. The r e s u l t s showed a 50-fold v a r i a t i o n i n the concentration of 2+ 2 + Fe and a 6-fold v a r i a t i o n i n the concentration of Cu among / 24. 2 + the media (Table V I I ) . The Fe concentration found i n 0.5M NaCl by the a n a l y t i c a l method used showed good agreement with that calculated from the data of impurities l i s t e d on the reagent bottles (Table V I I ) . The involvement of trace-metal ions i n promoting the dark oxidation of oxylumazine was confirmed by adding EDTA to synthetic seawater and buffered 0.5M NaCl. In both cases, the reaction was completely abolished (Table V I I I and F i g . 3). Reversal of the i n h i b i t i o n was attempted by 2 + adding Fe at a concentration s u f f i c i e n t to neu t r a l i z e the EDTA, 2 + but no reaction was observed; however s i m i l a r a ddition of Cu caused the expected reversal (Table V I I I ) . The reversed reactions showed the same stoichiometric differences between seawater and 2 + NaCl media as the controls without added EDTA and Cu , but the rates were s i g n i f i c a n t l y d i f f e r e n t . In addition to the trace-metal ion requirement, an absolute requirement f o r NaCl concentra-t i o n for the reaction was demonstrated by t e s t i n g the effe c t of 2+ 2 + Fe , with and without Cu , on oxylumazine i n pH 8 buffer with-out NaCl, when no reaction was observed (Table VIII and F i g . 3). Xanthopterin, s t r u c t u r a l l y analogus to oxylumazine, under-went zero-order dark reactions i n both seawater and buffer solutions (Table IV). In each case, with the exception of pH 10.0 buffer and ion mix, chromatographic analysis i n solvent system (I) revealed the presence of leucopterin (xanthopterin R f = 0.34; leucopterin i n seawater R f = 0.09 or i n buffer solutions R f = 0.11). Co-chromatography i n solvent system (I) and a spectral comparison of the eluted leucopterin band with authentic leucopterin confirmed the i d e n t i t y of t h i s compound. The rates f o r the disappearance of xanthopterin and the appearance of leucopterin i n the various media (Table IX) were determined from the concentration versus time curves. A direc t comparison of the xanthopterin disappearance rates to the leucopterin appearance rates was not considered v a l i d f o r estimating the reaction stoichiometry, because of the simulta-neous further reaction of part of the leucopterin produced (Table IV). When t h i s simultaneous leucopterin disappearance is taken into account ( f o r d e t a i l s , see Table IX), a 1:1 stoichiometric conversion of xanthopterin to leucopterin i s estimated f o r a l l the media tested. The addition of SDTA (2.5;uM) to xanthopterin i n 0.05M phosphate buffer (pH 8.0) had no e f f e c t on the dark reaction. Chelation Dioxylumazine showed considerable difference i n i t s spectrum i n seawater ( F i g . 2) from that i n 1.0M NaCl ( F i g . 4 ) . This difference appeared to be due to the presence of divalent 2+ 2+ cations i n the seawater. When the divalent cations (Ca , Mg and Sr" ) were not present i n synthetic seawater (modified seawater F i g . 7), the r e s u l t i n g spectrum was i d e n t i c a l to the 1.0M NaCl and the pH 8.3 T r i s buffer spectra (Fig.'7). Also, the addition 2+ 2+ n „ of Ca or Mg to pH 8.3 T r i s b u f f e r at the concentration found i n synthetic seawater a l t e r e d the b u f f e r spectrum p r o f i l e to that of the pteri d i n e i n seawater ( F i g . 7). Leucopterin, s t r u c t u r a l l y analogous to dioxylumazine, was found to be s i m i l a r l y a f f e c t e d by the presence of divalent 2+ 2+ 2+ cations. The spectrum with Ca , Mg , and Sr omitted from 26. seawater (modified seawater) agreed with the pH 8.3 T r i s buffer 2+ 2 + spectrum (Fig. 7). When Ca or Kg was added to pH 8.3 T r i s buffer, the spectrum was altered to a form i d e n t i c a l with that of the seawater spectrum ( F i g . 7). Also, the spectrum of , 2+ 2+x leucopterin i n the ion mix (Ca and Mg ) medium and i n seawater were i d e n t i c a l . For a l l the other pteridines studied where the Cg- or Cy-2+ 2 + or both hydroxyl groups were absent, no e f f e c t of Ca , Mg , or 2 + Sr i n seawater was observed. This suggested that a combina-t i o n of Cg- and Cy-hydroxyl groups and the divalent cations of seawater were responsible f o r the s p e c t r a l changes observed. This i n d i c a t i o n was s i m i l a r l y tested with a p t e r i d i n e lacking a l l substituents excepting the Cg, Cy-hydroxyl groups v i z 6,7-dihydroxypteridine. The e f f e c t of divalent cations on i t s spectrum was i d e n t i c a l with the observations f o r dioxylumazine and leucopterin. The spectrum i n modified seawater agreed with 2+ 2 + the T r i s buffer spectrum and the additi o n of Ca or Mg to the T r i s buffer a l t e r e d the spectrum to the form observed i n seawater (Figs. 7 and 8). An in t e r e s t i n g outcome of these observations was the close s i m i l a r i t y of the spectrum of each Cg, Cy-dihydroxypteridine i n seawater to i t s corresponding spectrum i n pH 10 buffer ( F i g . 7), suggesting that the complexation of these pteridines with the seawater divalent cations produces molecular species c l o s e l y comparable to those of the uncomplexed pteridines at pH 10. 27. Photolysis The Light Source The spectrum of the cool-white fluorescent lamps used i s presented i n Figure 9. Two kinds of spectral energy emission were observed, the continuous spectrum emitted by the fluorescent powder and the narrow bands of energy given out by the mercury arc i t s e l f at (309), 365.0, 404.7, 435.8, 546.1 and 578.Onm v (Sylvania B u l l e t i n , see Bibliography). The absorption spectrum (Fig. 10) of the glass from the photolysis f l a s k s , 0.17 + 0.02mm i n thickness, appeared to have l i t t l e e f f e c t on the spectrum of the cool^-white fluorescent lamps. Only i n the region of 300-350nm without the glass (Fig. 9, insert B) when compared to the same region with the photolysis f l a s k glass ( F i g . 9, i n s e r t C) was any effect observed. The reduction i n the 309nm mercury band was the most s t r i k i n g modification, but complete absorption of a l l wavelengths between 220-300nm also occurred. In the fluorescent lamp spectrum, only about 1.7% of the t o t a l spectral energy was below 380nm (Sylvania b u l l e t i n ) , so that the flasks appeared to have very l i t t l e e f fect on the t o t a l s p e c t r a l energy reaching the pteridines and purines. The major portion of the t o t a l s p e c tral energy (89.7%) was between 380-630nm, with 8.6% between 630-700nm. (These percentage values were calculated from data supplied i n the Sylvania b u l l e t i n and included the contribution from the mercury bands). The measured i l l u m i n a t i o n i n t e n s i t y at the l e v e l of the reaction flasks was 195 i 20 foot-candles (Weston Foot-candle Light Meter) or (6.0 + 0.3) x 10 erg/cm -sec (Kettering Radiometer). 28. Light Reactions - 'Photolysis' The r e s u l t s from the photo l y t i c reactions of pteridines are presented i n Tables X to XV. On considering the data^various rea c t i o n rate patterns i n seawater appeared to be r e l a t e d to the pterid i n e structure as follows: (A) A l l the Cg, Cy-hydroxylated pteridines (Tables X-XII, IV-V) underwent f i r s t - o r d e r reactions; however, oxylumazine and xanthopterin showed the highest reaction rates, about f o u r - f o l d greater than those observed f o r dioxy-lumazine, isoxanthopterin and leucopterin. (B) Lumazine and p t e r i n (Table XIIJ.) underwent slow zero-order reactions; however, the reaction rates f o r pt e r i n were about f o u r - f o l d greater than those observed for lumazine. In the buffer solutions, several relationships between pteridines and reaction rate patterns were observed. On consider-ing the pH p r o f i l e s , the following reaction patterns were noted: (A) Dioxylumazine ( F i g . 11) and leucopterin ( F i g . 12) showed very ra p i d reaction rate increase with pH i n the alk a l i n e range. The react i o n rates for both pteridines were f i r s t - o r d e r at pH 10.0 (Table X and XII) and zero-order at a l l other pH values. (B) Oxylumazine (Fig.' 13) and xanthopterin ( F i g . 14) showed very rapid reaction rate increase with pH i n the a c i d i c range. The reaction rate f o r both pteridines was zero-order at pK 4.0, second-order at pH 5.0 for xanthopterin only (Table XV), and f i r s t - o r d e r for both pteridines at a l l other pH values. (C) Isoxanthopterin (Fig. 15) showed a rapid reaction rate decrease with pH increase i n the a c i d i c range up to pH 6.0-7.0 and then a rap i d rate increase with pH i n the a l k a l i n e range. The reaction rates were zero-order at pH 6.0-7.0 and f i r s t - o r d e r at a l l other pH values studied. 29. (D) Lumazine and p t e r i n (Table XIII) underwent zero-order reactions at pH 8.0 and pH 10.0. The rates were observed to increase towards a l k a l i n e pH values, with the rates observed f o r fceiVig Those p t e r i n about t h r e e - f o l d greater than observed f o r lumazine. A A In c o r r e l a t i n g pH rate patterns with seawater rate patterns^ i t appeared that, with the exception of dioxylumazine (Fig. 11; Table X) and leucopterin ( F i g . 12; Table XII), the other pteridines showed seawater rates c l o s e l y p a r a l l e l i n g the pH 8.0 buffer r a t e . Dioxylumazine and leucopterin were exceptional i n showing f i r s t -order reaction and gre a t l y enhanced rate i n seawater as opposed to zero-order reaction i n buffer of the corresponding pH; the reasons f o r t h i s difference w i l l be discussed l a t e r . The f i n a l changes i n the absorption spectra from the photolysis of dioxylumazine ( F i g . 16), isoxanthopterin (Fig. 17), leucopterin ( F i g . 18), oxylumazine (Fig. 19), and xanthopterin ( F i g . 20) i n seawater indicated the ultimate formation of u n i d e n t i f i e d f a r UV-absorbing, degradation products from the marked increase i n r a t i o of absorption i n the 205-250nm region r e l a t i v e to that i n the 300-400nm region of the f i n a l spectrum. When the progress i n the s p e c t r a l changes during photolysis of these pteridines was c l o s e l y followed, no cle a r evidence of the formation of other p t e r i d i n e intermediates was obtained excepting the cases of oxylumazine and xanthopterin described i n the succeeding paragraphs. The following c r i t e r i a were used i n deducing the formation of pteridine intermediates during photolysis: ( i ) the progressive appearance of new absorption maxima or displacement of ex i s t i n g maxima i n the 300-400nm region, 30. ( i i ) k i n e t i c considerations of the transient concentrations of the o r i g i n a l p t e r i d i n e monitored at two pH's during the photolysis. The s p e c t r a l changes observed with lumazine ( F i g . 2l) and p t e r i n ( F i g . 22) were so s l i g h t as to be of doubtful s i g n i f i c a n c e and i t appears that these pteridines may be regarded as quite stable under the photolysis conditions used. When the progress i n the sp e c t r a l changes during photolysis of oxylumazine was c l o s e l y followed, the absorption p r o f i l e c h a r a c t e r i s t i c of dioxylumazine i n the 3l0-360nm region was barely discerned a f t e r 200-300 hour reaction i n seawater ( F i g . 19) or i n pH 8.0 buffer. This appearance of the c h a r a c t e r i s t i c absorption peaks of dioxylumazine was best evident from the slower, more prolonged p h o t o l y t i c r e a c t i o n of oxylumazine i n pH 7.0 buffer ( F i g . 23) but was altogether absent from the photolysis tested at pH 10.0. The formation of dioxylumazine as an intermediate/ of the photolysis of oxylumazine i n the various media, excepting pl-l 10.0 buffer, was v e r i f i e d by paper chromato-graphy of the reaction products i n solvent system (V), with authentic oxylumazine and dioxylumazine as standards. In t h i s solvent system, the oxylumazine standard showed the same Rf value (0.19) i r r e s p e c t i v e of the medium (seawater or a buffer) i n which t h i s standard was dissolved. However, the dioxylumazine standard showed one R^  value (0.07) from seawater s o l u t i o n and another R^  value (0.13) from a buff e r s o l u t i o n ( i r r e s p e c t i v e of pH); t h i s odd behavior of dioxylumazine i s a t t r i b u t e d to i t s complexation with the divalent cations of seawater producing a chelated species of lower R^  value chromatographically d i s t i n c t from the uncomplexed species obtained i n a buffer s o l u t i o n . The chromatography of the oxylumazine photolysis products i n v a r i a b l y showed a pteridine co-chromatographing with authentic dioxylumazine, i n additi o n to unreacted oxylumazine. The i d e n t i t y of t h i s p t e r i d i n e intermediate with dioxylumazine was further confirmed by spectral comparison a f t e r e l u t i n g i t from the paper chromatograms. In no case was the concentration of dioxylumazine formed very substantial ( F i g s . 24-27). The photo-l y t i c breakdown of authentic dioxylumazine i n seawater and pH 8.0 buffer 7known from the r e s u l t s shown i n Table X, would be expected to off s e t i t s accumulation as an intermediate i n these media, but i n pH 7.0, 5.4, and 4.0 buffers t h i s accumulation would be favoured to show apparent s t a b i l i t y or even increase i n concentration. In the pH 7.0 buffer, the spectrum of the oxylumazine photolysis product at 355 hours of reac t i o n ( Fig. 23) showed considerably high absorbance i n the 205-250nm range r e l a t i v e to the i n t e n s i t y of the dioxylumazine peaks i n the 3l0-360nm range ( l i t t l e i f any oxylumazine was l e f t at 355 hours, see F i g . 26), indi c a t i n g that other f a r UV-absorbing intermediates or products were being simultaneously formed. Si m i l a r observa-tions of such intermediates or end products i n the f a r UV-region were made with the oxylumazine photolysis reactions i n other media where indications of dioxylumazine intermediate formation were obtained. When the progress i n the sp e c t r a l changes during photolysis of xanthopterin was c l o s e l y followed, the absorption p r o f i l e c h a r a c t e r i s t i c of leucopterin i n the 280-380nm region was barely discerned a f t e r 200-300 hour reaction i n seawater ( F i g . 20) and i n pH 8.0 buf f e r . This appearance of the c h a r a c t e r i s t i c 32. absorption peaks of leiacopterin was best evident from the slower, more prolonged photolytic reaction of xanthopterin i n pH 7.0 buffer ( F i g . 23) (cf. F i g . 29, authentic leucopterin at pH 1.0 f o r an approximate s p e c t r a l p r o f i l e of authentic leucop-t e r i n at pH 7.0, als o values i n Table I I ) . The c h a r a c t e r i s t i c peaks of leucopterin were absent from the photolysis tested at pH 10.0. The formation of leucopterin as an intermediate of the photolysis of xanthopterin i n the various media, excepting pH 10.0 buffer, was v e r i f i e d by paper chromatography of the reaction products i n solvent system ( i ) , with authentic xanthopterin and leucopterin as standards. In t h i s solvent system, the xanthop-t e r i n standard showed the same value (0.33) i r r e s p e c t i v e of the medium (seawater or a buffer) i n which t h i s standard was dissolved. However, the leucopterin standard showed one R^ value (0.09) from seawater s o l u t i o n and another value (0.12) from a buffer s o l u t i o n ( i r r e s p e c t i v e of pH); t h i s odd behavior of leucopterin, s i m i l a r to that of dioxylumazine, i s again a t t r i b u t e d to i t s complexation with the divalent cations of seawater producing a chelated species of lower R^  value chromato-gr a p h i c a l l y d i s t i n c t from the uncomplexed species obtained i n a buffer s o l u t i o n . The chromatography of the xanthopterin photolysis products i n v a r i a b l y showed a pteridine co-chromatographing with authentic leucopterin, i n addition to unreacted xanthopterin. The i d e n t i t y of t h i s pteridine intermediate with leucopterin was further confirmed by spectral comparison a f t e r e l u t i n g i t from the paper chromatograms. The concentrations of leucopterin and xanthopterin versus time i n the various media are presented i n Figures 30 to 34. The photolytic breakdown of authentic leucop-33. t e r i n in seawater, ion mix medium, and pH 8.0 buffer known from the results shown i n Table XII, would be expected to o f f s e t i t s accumulation as an intermediate in, these media but i n pH 5.0, and 4.0 buffers t h i s accumulation would be favoured to show apparent s t a b i l i t y or even increase i n concentration. In the pH 7.0 buffer the spectrum of xanthopterin photolysis product at 740 hours of reaction (Fig. 28) showed considerably higher absorbance i n the 205-250nm range r e l a t i v e to the i n t e n s i t y of the leucopterin peak at 299nm (very l i t t l e leucopterin was l e f t other at 740 hours, see F i g . 34), i n d i c a t i n g t h a t ^ f a r UV-absorbing intermediates or end products were being formed. S i m i l a r observations of such intermediates or end products i n the f a r UV-region were made with the xanthopterin photolysis reactions i n other media where indications of leucopterin intermediate formation were obtained. Purines Dark Reactions The results from the dark reaction of purines i n NSM-medium are presented i n Table XVI, Figures 36-40. The data show that the c h a r a c t e r i s t i c absorption maxima of a l l the purines were not alt e r e d by prolonged storage i n darkness but that temporal changes i n the absorbance at these maxima were observed. Adenine, guanine, hypoxanthine, and xanthine a l l increased i n absorbance over the time period of study (Fig. 36). In each case a very rapid increase i n absorbance occurred between 0 to about 250 hours for which no rate constants were calcul a t e d (except for guanine) because i n s u f f i c i e n t readings were taken to conclusively 34. say i f the increase was zero- or f i r s t - o r d e r i n k i n e t i c s . A f t e r t h i s i n i t i a l rapid increase, two d i f f e r e n t patterns were observed over the remaining time period of study: (A) the absorbance of hypoxanthine and xanthine remained constant and (B) the absorbance of adenine and guanine increased very slowly by zero-order k i n e t i c s . The o v e r a l l increase i n absorption of adenine, guanine, hypoxanthine, and xanthine (Figs. 37-38) was most marked i n the major peaks (above 225nm) and i n the 300-350nm spec t r a l region. U r i c acid unlike the other purines,underwent a zero order decomposition reaction i n the dark ( F i g . 39). The progressive changes i n the absorption spectrum during decomposition ( F i g . 40) do not show any clear evidence of spec t r o s c o p i c a l l y i d e n t i f i a b l e intermediates; however, there appears to be a trend tov/ards an increase i n the 236nm peak i n t e n s i t y r e l a t i v e to the 292nm peak, possibly i n d i c a t i n g the formation of some far UV-absorbing products. Light Reactions - 'Photolysis' The results from the photol y t i c reactions of purines i n NSM-medium are presented i n Table XVII, Figures 36-40. Sim i l a r to the dark reactions, an increase i n absorbance at the characteri-s t i c maxima was observed for adenine, guanine, and hypoxanthine (F i g . 36). Adenine and hypoxanthine showed an i n i t i a l rapid change (increase and decrease respectively) during the f i r s t 50 hours, then a zero-order increase i n absorbance over the remaining time period of study, whilst guanine showed a zero-order rate of increase i n absorbance over the complete period. The o v e r a l l increase i n absorption of adenine and guanine ( F i g . 37) was most marked i n the major peaks (above 225nm) and i n the 300-350nm sp e c t r a l region. In the f i n a l spectrum of hypoxanthine (Fig. 33) no o v e r a l l change i s apparent i n the major peak because the i n i t i a l decrease was equal to the subsequent increase i n absorbance ( F i g . 36). Ur i c acid and xanthine both underwent a decomposition reaction i n NSM-medium under cool-white fluorescent lamps (Table XVII). Xanthine showed a slow zero-order reaction (F i g . 36) while u r i c acid underwent a fa s t e r f i r s t - o r d e r reaction ( F i g . 39). The s p e c t r a l changes i n xanthine ( F i g . 38) showed decreases i n the major peaks (above 225nm) and a small increase i n the 3l0-350nm region. No cl e a r evidence of spect r o s c o p i c a l l y i d e n t i f i a b l e intermediates or products was obtained from the reaction of e i t h e r xanthine ( F i g . 38) or u r i c acid ( F i g . 40). 36. DISCUSSION. Theor e t i c a l Considerations The s t a b i l i t y of pteridines or purines under l i g h t and dark conditions i n seawater has not been previously studied. The r e s u l t s of t h i s i n v e s t i g a t i o n have shown that the pteridines were generally unstable while the purines were stable i n sea-water. This general difference i n r e a c t i v i t y of these two types of nitrogen-containing compounds r e f l e c t e d the chemical basis of the heteroaromatic class to which they belong. These two classes are: (I.) ring-systems i n which the carbon atoms have a deficiency of pi-electrons ( p t e r i d i n e s ) , and (II) r i n g -systems with an excess of pi-electrons (purines). T h e o r e t i c a l considerations of the chemistry of aromatic compounds have led to the r e a l i z a t i o n that t h e i r s t a b i l i t y is governed by t h e i r content of pi-electrons and that the pi-excessive systems are more stable than p i - d e f i c i e n t ones (Albert, 1959). Almost a l l p i - d e f i c i e n t N-heteroaromatics are known to be six-membered ring-systems (Albert, 1959), containing the nitrogen atom i n a setting (=CH-N=CH-) that makes i t e l e c t r o n - a t t r a c t i n g , whereas the pi-excessive heteroaromatics contain a nitrogen atom i n an electron-releasing s e t t i n g (=CH-NH-CH=), generally i n a five-membered ring. Pteridines In p i - d e f i c i e n t systems, the e l e c t r o n - a t t r a c t i n g e f f e c t confers a p o s i t i v e charge upon the other r i n g atoms. This causes a p o l a r i z a t i o n of the molecule and, with fused-ring systems, the most po lar r ing becomes the most vulnerable to f i s s i o n . When more than one r i n g - n i t r o g e n is present , the fur ther loss of s t a b i l i t y i s seen i n the greater tendency to h y d r o l y t i c degrada-t i o n . A l so , the presence of a d d i t i o n a l n i trogen atoms i n a neighbouring r i n g inf luences the e l e c t r o n d i s t r i b u t i o n wi th in the molecule and thus the s t a b i l i t y of the e n t i r e r ing system. A rough index of the i n s t a b i l i t y i s a measure of the N:C r a t i o of the r ing atoms. As the N:C r a t i o increases , s t a b i l i t y decreases. For example, p t e r i d i n e with a J*:C r a t i o of 0.7:1 i s unstable to co ld aqueous (IN) a c i d or a l k a l i , being degraded to 2-aminopyrazine-3-aldehyde ( A l b e r t , Brown, and Wood, 1956; A l b e r t , and Yamamoto, 1963a), whi l s t q u i n o l i n e , with a N:C r a t i o of 0 .1:1 , i s s table i n hot concentrated a c i d or base (Alber t , 1959). Dark Reactions Inser t ion of hydroxyl - or amino-groups ( e l e c t r o n - r e l e a s i n g groups) i n t o p i - d e f i c i e n t systems reduces t h e i r i n s t a b i l i t y (A lber t , 1959). With the presence of two or more hydroxy l -groups i n p t e r i d i n e , the i n s t a b i l i t y to a c i d and a l k a l i i s l a r g e l y overcome ( A l b e r t , Brown, and Cheeseman, 1952b). The resu l t s of the present study have shown that the s t a b i l i t y of 2 , 4 -d i subs t i tu ted pter id ines i n seawater under dark condit ions i s r e l a t e d not only to the number of subst i tuents but a l so to the p o s i t i o n of the subst i tuents i n the pyrazine r i n g . When no such subst i tuents were present as i n lumazine and p t e r i n , or a 7-hydroxyl group as i n i soxanthopter in , no reac t ion was detected, i n d i c a t i n g h igh s t a b i l i t y from such s t r u c t u r e s . On the other 38. hand, the presence of a 6-hydroxyl group as i n oxylumazine and xanthopter in appeared to markedly reduce s t a b i l i t y r e s u l t i n g i n the greatest r e a c t i v i t y of a l l the p ter id ines t e s ted . The simultaneous presence of a 7-hydroxyl group, as i n dioxylumazine and l eucopter in , appeared to decrease th i s r e a c t i v i t y of 6-hydroxypteridines and to s t a b i l i z e them i n seawater. No general c o r r e l a t i o n s h i p between the number of subst i tuents and t h e i r p o s i t i o n i n r e l a t i o n to r e a c t i v i t y was observed i n the present study with the p ter id ines i n the various buf fer so lut ions tes ted . However, the pH behaviour of i n d i v i d u a l p ter id ines showed close correspondence with r e l a t e d propert ies reported i n the l i t e r a t u r e , where a v a l i d comparison was p o s s i b l e , s ince the s t a b i l i t y of the p ter id ines r e l a t i v e to pH has not been p r e v i o u s l y examined i n any systematic fashion. The buf fer s o l u -t i o n tes t s showed dioxylumazine to be s table at a c i d i c pH and unstable at a l k a l i n e pK, i n close agreement with s i m i l a r observations of A l b e r t , Brown, and Cheeseman (1952b). Lumazine was observed i n t h i s study to be s table i n pH 10.0 and 8.0 buf fers , whi l s t i t has been reported to decompose i n 10'M-NaOH at 110°C ( A l b e r t , Frown, and Cheeseman, 1952b). Xanthopterin showed considerable r e a c t i v i t y i n a c i d i c and basic b u f f e r s , wh i l s t i t was reported to be unaffected by b o i l i n g HCl and s l i g h t l y af fected by b o i l i n g barium hydroxide (Wieland, and Schopf, 1925; Wieland, and Purrmann, 1940). Leucopterin was observed to be s l i g h t l y a f fec ted i n the a c i d i c b u f f e r s , which was a l so reported to be the case i n hot concentrated I^SOA (Wieland et a l . , 1933). The observed s i m i l a r i t y of the seawater dark r e a c t i o n rates of a l l the p ter id ines t es ted (excepting oxylumazine) to the rates i n the buffers at pH 8 . 0 -S .3 ( c l o s e l y corresponding to seawater pH) suggests that the seawater s a l t s may have l i t t l e chemical e f f ec t on the p ter id ines and that the dark react ions i n sea-water were a consequence of i t s pH. On the other hand, oxy-lumazine was s table at a l l pH values s tudied but remarkably unstable i n seawater, i n d i c a t i n g a strong chemical e f f ec t of the s a l t concentrat ion of seawater on i t s r e a c t i v i t y . This was confirmed by the studies using NaCl so lut ions of d i f f e r e n t s trengths , where a minimal s a l t concentrat ion was found to be required for detectable r e a c t i o n . However, i n a d d i t i o n to th i s s a l t requirement, a further requirement for a r e a d i l y reducing 2 + trace-meta l i o n , such as Cu , was e s t a b l i s h e d . The importance 2 + of Cu to the oxylumazine reac t ion i n seawater and i n NaCl so lut ions was confirmed by the i n h i b i t i o n of the reac t ion by EDTA. Upon r e v e r s a l of the i n h i b i t i o n by c o n t r o l l e d a d d i t i o n 2 + of Cu , the d i f ference i n reac t ion rates from the o r i g i n a l (EDTA untreated) rates , suggested that the r e a c t i o n rate was 2 + dependent upon the Cu concentrat ion . Since no reac t ion was 2 + observed with Cu alone i n the absence of NaCl at the pH of seawater, i t was concluded that the presence of minimal 2+ concentrations of both NaCl and Cu were required for the r e a c t i o n . In t h i s connection, i t i s pointed out that the minimal NaCl concentrat ion required may be lower than the a c t u a l r e s u l t reported i n Figure 3, s ince t h i s tes t has ignored the 2 + minimal Cu concentrat ion required for the r e a c t i o n . 40. The formation of dioxylumazine as an intermediate or product of the react ion of oxylumazine was confirmed i n both seawater and NaCl s o l u t i o n s ; however, a d i f f erence i n reac t ion s to ichiometry was observed between the two media. Whereas i n the NaCl so lut ions a 1 : 1 conversion of oxylumazine to dioxy-lumazine was e s tab l i shed , the seawater r e a c t i o n measurements i n d i c a t e d a 2 : 1 conversion, i n d i c a t i n g the formation of a d d i t i o n a l product(s) q u a n t i t a t i v e l y equivalent to dioxylumazine. This a d d i t i o n a l u n i d e n t i f i e d reac t ion must be a t t r i b u t e d to the a c t i o n of ions other than NaCl i n the seawater. The 1 : 1 s to ichiometry of the reac t ion i n NaCl solutions was not changed when KC1 replaced N a C l , while the 2 : 1 s to ichiometry of the reac t ion i n seawater / 2+ was a l so unaffected by the omission of d iva lent cat ions (Ca , 2+ 2 + Mg , Sr ) from the seawater. It thus appeared that the s t o i c h i o m e t r i c d i f ference was not due to the major cations of seawater. The 1 : 1 s to ichiometry of the r e a c t i o n i n NaCl s o l u t i o n 2-was a l so not a l t e r e d by the i n d i v i d u a l major anions (SO^ , E r , 3- -POg , HCOg , F ) of seawater or random combinations of pa irs of these anions , although the r e a c t i o n rates were markedly a l t e r e d by some of the ca t ions . Since a 2 : 1 s to ichiometry was / 2 + obtained with modified seawater lacking d iva lent cat ions (Ca , 2+ 2 + Mg , 3 r ) but containing a l l the above mentioned anions, i t appeared that the d i f ference i n s to ichiometry between the r e a c t i o n rates i n seawater and NaCl so lut ions may be due to a unique combination of the t o t a l seawater anions which could not be further defined i n t h i s study. 41. The formation of dioxylumazine from oxylumazine and of l e u c o p t e r i n from x a n t h o p t e r i n i n the dark (and a l s o i n the l i g h t ) r e a c t i o n s were unexpected obs e r v a t i o n s . I t i s reported t h a t dioxylumazine and l e u c o p t e r i n are e n z y m a t i c a l l y produced by the o x i d a t i o n of oxylumazine and x a n t h o p t e r i n r e s p e c t i v e l y , by xanthine oxidase (Bergmann, and Kwietny, 1958, 1959; Schou, 1950). This enzymatic o x i d a t i o n i s b e l i e v e d t o take p l a c e i n two s t e p s : ( i ) h y d r a t i o n of the 7,8-double bond and ( i i ) dehydrogenation of t h i s hydrated-bond (]?ergmann, and Kwietny, 1959; McCormack, and V a l e r i n o , 1970), both these r e a c t i o n s being assumed t o occur a f t e r the s u b s t r a t e i s adsorbed onto the enzyme s u r f a c e . Mow oxylumazine and x a n t h o p t e r i n are known t o spontaneously undergo v a r y i n g degrees of non-enzymatic h y d r a t i o n across the 7,8-double bond (Inoue and P e r r i n , 1963a; A l b e r t , 1969) i n aqueous s o l u t i o n i n the pH range 1-10; whereas the non-hydrated molecular species i s r e p o r t e d t o be the only s u b s t r a t e f o r enzyme a t t a c k (Schou, 1950; Inoue, and F e r r i n , 1962). I t appears t h a t , c h e m i c a l l y speaking, both hydrated and non-hydrated species can undergo o x i d a t i o n , depending on the reagent used. The c a t a l y t i c o x i d a t i o n of the non-hydrated x a n t h o p t e r i n molecule t o l e u c o p t e r i n i n a c e t i c a c i d w i t h molecxalar oxygen over platinum has been reporte d O'ieland, and Purrmann, 1940; C'Dell et a l . , 1947). Hydrogen p e r i o x i d e has been shown t o o x i d i z e the hydrated x a n t h o p t e r i n t o l e u c o p t e r i n , a p p a r e n t l y by a dehydroga-t i o n mechanism (Wieland, and Purrmann, 1939; A l b e r t , Frown, and Cheeseman, 1952a). O x i d a t i o n of hydrated p t e r i d i n e s by another m i l d o x i d a t i v e reagent (aqueous potassium permanganate at room 42. temperature) is known and is u t i l i z e d to determine the s i t e of hydrat ion i n p ter id ines and r e l a t e d h e t e r o c y c l i c systems (see Jacobsen, 1966; Brown, and Mason, 1956). Rembold (1970) reported that 7 , 8-dihydrolurnazine and 7 , 3 -d ihydropter in which both hydrate across the 5,6-double bond, were ox id ized by a i r to 7,8-dihydro-6-hydroxylumazine and 7 ,8 -d ihydroxanthopter in while i n buffered media. The case of hydrat ion i n an apparently f u l l y aromatic molecule i s not immediately evident , but may l i e i n the h igh e l e c t r o n - a f f i n i t y of each double-bonded ni trogen atom ( A l b e r t , 1964). The presence of s evera l doubly-bonded n i trogen atoms i n a heteroaromatic nucleus ( e s p e c i a l l y i f they are placed 1,3 to one another so that t h e i r separate e f fects are add i t ive ) tends to deplete the p i - e l e c t r o n layer of the nucleus so s trong ly that normal aromatic activit}' - d isappears . As a r e s u l t , a h i g h l y p o l a r i z e d i s o l a t e d double-bond i s exposed, and becomes suscept ib le to a t tack from n u c l e o p h i l i c reagents. Thus water (a weak nuc leophi le ) adds across the 7,8-double bond of 6-hydroxylated p t e r i d i n e s . The presence of the 6-hydroxyl group g r e a t l y ac t ivates the 7,8-double bond so that t h i s s i t e i s favoured both k i . n e t i c a l l y and thermodynamically for hydrat ion ( A l be r t , Baterham, and McCormack, 1966). A l s o , the hydrat ion across the 7,8-double bond i s promoted by d iva lent metal ions by c h e l a t i o n with them as shown i n Figure 47B (Rowan et a l . , 1961). The hydrat ion and dehydration k i n e t i c s of xanthopterin over the pH range of 3.7-11.8 has been studied by Inoue, and P e r r i n (1963a). The percentage of hydrated and non-hydrated xanthopterin c a l c u l a t e d from t h e i r data i s presented i n Figure 41. Below pH 43. 3.7, no k i n e t i c data was presented by these authors and the curve ( ) i n t h i s r e g i o n has been e x t r a p o l a t e d from the p e r t i n e n t xanthine oxidase s t u d i e s r e p o r t e d by Schou (1950). To date, the hy d r a t i o n - d e h y d r a t i o n k i n e t i c s of oxylumazine have not been reported. Unpublished i n f o r m a t i o n from P r o f e s s o r A l b e r t (1969) has i n d i c a t e d t h a t ( i ) oxylumazine enters i n t o a slow e q u i l i b r i u m w i t h i t s 7,8-covalent hydrate, of which a few parts percent may be present at e q u i l i b r i u m , but i s not so prone t o h y d r a t i o n as i s x a n t h o p t e r i n , and ( i i ) the appearance of a shoulder i n the 300nm s p e c t r a l r e g i o n (see F i g . 5, oxylumazine at pH 1.0) of the n e u t r a l species and c a t i o n i s i n d i c a t i v e of the formation of such a hydrate. In Figure 42, an exaggerated, h y p o t h e t i c a l curve f o r the precentage h y d r a t i o n of oxylumazine, based on t h i s unpublished i n f o r m a t i o n , i s presented. The o x i d a t i o n of x a n t h o p t e r i n and oxylumazine may have i n v o l v e d e i t h e r the non-hydrated or hydrated molecular s p e c i e s . The m i l d c o n d i t i o n s r e q u i r e d f o r o x i d a t i o n of p t e r i d i n e s which form covalent hydrates, e s p e c i a l l y the a i r o x i d a t i o n of 7,8-dihydrolumazine and 7,8-dihydropterin t o 7,8-dihydro-6-hydroxyr lumazine and 7,8-dihydroxanthopterin i n b u f f e r e d media, suggests t h a t the hydrated species of x a n t h o p t e r i n and oxylumazine may be s i m i l a r l y dehydrogenated t o l e u c o p t e r i n and dioxylumazine r e s p e c t i v e l y . In t h i s connection, i t appears from the present s t u d i e s that pH as w e l l as s a l t c o n c e n t r a t i o n may have e f f e c t s on the o x i d a t i o n of 6-hydroxypteridines presumably v i a formation of t h e i r 7,8-covalent hydrates. X a n t h o p t e r i n and oxylumazine have shown some s t r i k i n g d i f f e r e n c e s i n t h e i r o x i d a t i o n p o t e n t i a l : 44. a) x a n t h o p t e r i n i s s 1 ow 1 y o x i d i z e d through zero-order r e a c t i o n  k i n e t i c s at extremely a c i d i c p'l (^4.0) and at a l k a l i n e pH (7.0-10.0), appears t o be u n a f f e c t e d at pH 5.0-6.0, and does not r e q u i r e the s a l t c o n c e n t r a t i o n nor the t r a c e - m e t a l i o n content of seawater f o r comparable o x i d a t i o n at the pl-l of seawater; b) oxylumazine i s not d e t e c t a b l y o x i d i z e d over the e n t i r e pH range (4.0-10.0) examined w i t h the b u f f e r s alone but undergoes r a p i d o x i d a t i o n through f i r s t - o r d e r r e a c t i o n k i n e t i c s w i t h the s a l t c o n c e n t r a t i o n and tra c e - m e t a l i o n content of seawater and shows a d e f i n i t e requirement f o r s a l t c o n c e n t r a t i o n as w e l l as 2+ t r a c e - m e t a l i o n (at l e a s t Cu ) f o r such o x i d a t i o n . The nature of these d i f f e r e n c e s r e f l e c t s fundamental d i f f e r e n c e s i n molecular p r o p e r t i e s d e s p i t e t h e i r o v e r a l l s t r u c t u r a l s i m i l a r i t y and a l s o r a i s e s the p r o b a b i l i t y of d i f f e r e n c e s i n t h e i r r e a c t i o n mechanisms. The i n e r t n e s s of oxylumazine over the pH range s t u d i e d i n the absence of s a l t ( s ) c o r r e l a t e s w e l l w i t h i t s known tendency f o r poor h y d r a t i o n and at the same time i n d i c a t e s that the non-hydrated species i s s t a b l e . The remarkable increase i n i t s r e a c t i v i t y w i t h the s a l t c o n c e n t r a t i o n of seawater may at l e a s t p a r t i a l l y be exp l a i n e d by a s i g n i f i c a n t i n c r e a s e i n i t s h y d r a t i o n e f f e c t e d by the s a l t c o n c e n t r a t i o n . This i s suggested by s p e c t r o s c o p i c i n d i c a t i o n s of d e t e c t a b l e formation of the hydrated species soon a f t e r d i s s o l u t i o n of oxylumazine i n seawater or i n NaCl s o l u t i o n s but not i n pH 8.0 b u f f e r . A comparison of i t s pH 8.0 spectrum ( F i g . 5) w i t h the zero-hour s p e c t r a i n seawater ( F i g . l ) and i n NaCl s o l u t i o n s ( F i g . 4) 45. shows s i g n i f i c a n t i n c r e a s e i n a b s o r p t i o n i n t e n s i t y i n the 300nm r e g i o n (of the l a t t e r two spectra) accompanied by the appearance of an i n f l e c t i o n , which i s i n d i c a t i v e of the formation of a 7,3-covalent hydrate i n seawater and s a l t s o l u t i o n s ( A l b e r t , 1969). In c o n t r a s t t o the pH-inertness of oxylumazine, x a n t h o p t e r i n was r e a c t i v e at a l l pH values examined (excepting pH 5.0), which r e a c t i v i t y was not s i g n i f i c a n t l y a f f e c t e d by the s a l t s of seawater, and t h i s p r o p e r t y of x a n t h o p t e r i n c o r r e l a t e s w e l l w i t h i t s known tendency f o r ready h y d r a t i o n . The e x c e p t i o n a l i n e r t n e s s of x a n t h o p t e r i n at pi! 5.0 (where maximal h y d r a t i o n o c c u r s ) , i n c o n t r a s t t o i t s r e a c t i v i t y at pH's 4.0 and 7.0 (where h y d r a t i o n i s s t i l l maximal or c o n s i d e r a b l e ) , suggests however t h a t h y d r a t i o n alone i s not r e s p o n s i b l e f o r the observed r e a c t i v i t y and that an a d d i t i o n a l s t r u c t t . i r a l f a c t o r may be i n v o l v e d . In Figure 41, the v a r i o u s known molecular species of x a n t h o p t e r i n , as they may be expected t o occur over the pH range 1-12, are presented. At pH 5.0, about 99% i s present as the n e u t r a l species (hydrated plus non-hydrated) accompanied by 1% monoanion and v i r t u a l l y no c a t i o n , whereas at pH 4.0, w i t h about the same percentage of n e u t r a l s p e c i e s , the c a t i o n c o n c e n t r a t i o n has incre a s e d t o 0.37c and the anion c o n c e n t r a t i o n has diminished t o i n s i g n i f i c a n c e . Furthermore, the r e a c t i o n r a t e at pH 4.0 i s s l i g h t l y g r e a t e r than that at pH 7.0 where 577* i s present as the monoanion and no c a t i o n i s expected t o occur. These observations suggest t h a t , i n a d d i t i o n t o h y d r a t i o n , minimal concentrations of a n i o n i c or c a t i o n i c species (presumably hydrated) are r e q u i r e d f o r xanthop-t e r i n r e a c t i v i t y and th a t the c a t i o n i c species may promote t h i s 46. r e a c t i v i t y to a greater degree than the anionic species while the n e u t r a l species may be i n e r t . The d e f i n i t e requirement for a trace-metal d ivalent ca t ion / 2+x (such as Cu ) of the oxylumazine dark react ions i n seawater and NaCl so lut ions was demonstrated by the EDTA i n h i b i t i o n studies and 2 + r e v e r s a l of the i n h i b i t i o n by a d d i t i o n of Cu at a concentrat ion s u f f i c i e n t to n e u t r a l i z e the EDTA. Two poss ib le functions of 2+ / \ Cu i n the dark reac t ion are : ( i ) to act as a redox system i n the dehydrogenation r e a c t i o n , and/or ( i i ) to promote hydrat ion by formation of a che la t ion complex. The dehydrogenation of enzyme-hydrated p ter id ines by xanthine oxidase i s known to be 6+ 3 + f a c i l i t a t e d by heavy metal ions (Mo , Fe ) assoc iated with the f lav in-adenine d inuc leot ide (FAD) pros the t i c group (Avis et al., 1956; Fray 1963); the FAD p r o s t h e t i c group and the heavy metals have been shown to serve as a r e v e r s i b l e redox system, which accepts the hydrogens - or e lectrons - from the p ter id ines and transmits them further to oxygen. A s i m i l a r s i t u a t i o n to th i s i s reported to occur i n which cytochrome C -3 + cytochrome oxidase system invo lv ing Fe , f a c i l i t a t e d the ox idat ion of u r i c a c i d ( G r i f f i t h , 1952). G r i f f i t h s (1952) a l so observed 2 + that Cu , i n the absence of the cytochrome oxidase system, acted as the redox system i n the ox idat ion of u r i c a c i d . The 2 + other poss ib le r o l e of Cu may be i n the promotion of hydrat ion by formation of a d ivalent metal chelate ( F i g . 47B) (Rowan et a l . , 1961) but spectroscopic ind ica t ions suggest t h i s not to be the case. A comparison of the spectrum of oxylumazine at pH 8 .0 , 2 + with and without ( F i g . 5) Cu (at the concentrat ion observed i n 0.5M NaCl) showed no change i n the 300nm reg ion , which may be i n d i c a t i v e that formation of a 7,8-covalent hydrate i s not 47. enhanced t o any app r e c i a b l e e x t e n t , i f at a l l . A l s o , the 3OOnm-region sp e c t r a of oxylumazine i n seawater and 0.5M NaCl, w i t h and without EDTA, are i d e n t i c a l , suggesting that only the i o n i c s t r e n g t h of seawater and NaCl promote h y d r a t i o n and not ? + 2 + Cu . These observations suggest t h a t Cu may f u n c t i o n p r i m a r i l y as an e l e c t r o n " s h u t t l e r " , probably by complexing 2+ w i t h oxylumazine t o form a Cu -oxylumazine c h e l a t e . In c o n t r a s t t o oxylumazine, x a n t h o p t e r i n does not appear 2 + t o have a Cu requirement. The presence of EDTA d i d not i n h i b i t the x a n t h o p t e r i n dark r e a c t i o n i n pH 3.0 b u f f e r suggesting t h a t the dehydrogenation of hydrated x a n t h o p t e r i n t o l e u c o p t e r i n occurs d i r e c t l y w i t h molecular oxygen and does not r e q u i r e a redox system t o f a c i l i t a t e t h i s r e a c t i o n . T h i s d i f f e r e n c e i n r e a c t i o n mechanism probably r e f l e c t s the fundamental d i f f e r e n c e i n p t e r i d i n e s t r u c t u r e , a C2~amino group i n x a n t h o p t e r i n i n c o n t r a s t t o a C2-hydroxyl group i n oxylumazin The conversion of oxylumazine t o dioxylumazine i n NaCl s o l u t i o n s was s t o i c h i o m e t r i c a l l y 1:1 i n c o n t r a s t t o the 2:1 s t o i c h i o m e t r i c conversion i n seawater. With the requirement 2+ of the i o n i c c o n c e n t r a t i o n ( f o r h y d r a t i o n ) and Cu (as a redox s37-stem) the r e a c t i o n Scheme A ( F i g . 43) i s proposed f o r the 1:1 s t o i c h i o m e t r i c conversion i n NaCl s o l u t i o n s of oxylumazine t o dioxylumazine. The r e a c t i o n r a t e of oxylumazine (measured by i t s disappearance) i n seawater was approximately t w i c e as f a s t as t h a t i n NaCl s o l u t i o n of comparable i o n i c s t r e n g t h ; however the r a t e of formation of dioxylumazine was approximately equal i n two media. These ob s e r v a t i o n s , combined with the 2:1 48. s to ich iometry , ind icate that two simultaneous react ions are tak ing place i n the seawater, ( i ) the ox idat ion of a port ion of oxylumazine to dioxylumazine at a rate s i m i l a r to that observed i n NaCl s o l u t i o n , and ( i i ) the p a r a l l e l breakdown of another equal por t ion to u n i d e n t i f i e d product ( s ) . whereas both react ions 2 + requ ire Cu , the second reac t ion appears to require the combination and concentrat ion of the anions p e c u l i a r to seawater. It appears that the seawater anion ef fect a l t e r s the ox idat ion p o t e n t i a l of the hydrated oxylumazine i n a way whereby only h a l f of i t i s ox id ized v i a the Cu -complex and the other h a l f i s s imultaneously reduced and then degraded; i n t h i s case the 2 + Cu i s envisaged as accepting e lectrons from the f i r s t h a l f being ox id ized and regenerating a f t e r donating these e lectrons to the second h a l f being reduced. Since no evidence was obtained for the formation of a d ihydropter id ine product (unl ike the case of 6-hydroxypteridine; A l b e r t , 1955), i t appears that the second ha l f may be degraded at the time of or even p r i o r to the reduct ion s tep. Based on these cons iderat ions , r eac t ion Scheme B ( F i g . 43) i s proposed invoking the concerted ox idat ion-reduct ion of oxylumazine i n seawater r e s u l t i n g i n the 2:1 s to ichiometry observed. It w i l l be seen from th i s r e a c t i o n schane that the hydrated oxylumazine molecule being reduced is represented a r b i t r a r i l y as an apparently degraded p t e r i d i n e spec ies . This p o s t u l a t i o n is based on the known l a b i l i t y of the Cy-Ng bond of severa l p t e r i d i n e 7,8-covalent hydrates reported i n the l i t e r a t u r e (Rowan, and Wood, 1968). This l a b i l i t y i s p e c u l i a r to such hydrates i n that the Cy-Ng cleaved species i s a c t u a l l y 49. i n e q u i l i b r i u m w i t h the'Gy-Ng uncleaved species (Rowan, and Wood, 1968; or of N3-C4 bond, P e r r i n , 1962; Inoue and P e r r i n , 1963b). U n f o r t u n a t e l y , the degraded h a l f of the oxylumazine i n seawater could not be i d e n t i f i e d w i t h i n the scope of t h i s i n v e s t i g a t i o n , but i t appears from the s p e c t r o s c o p i c evidence t h a t i t may not be a pyrazine d e r i v a t i v e and t h e r e f o r e i t i s t e n t a t i v e l y i n f e r r e d t o be a p y r i m i d i n e d e r i v a t i v e . Such an i n f e r e n c e i s a l s o i n accord w i t h the Cy-Ng cleaved species p o s t u l a t e d i n Scheme B ( F i g . 43). The s t o i c h i o m e t r y of the r e a c t i o n s of x a n t h o p t e r i n i n seawater and b u f f e r s o l u t i o n s (without s a l t s ) was 1 x a n t h o p t e r i n : 1 l e u c o p t e r i n intermediate i n a l l cases where t h i s intermediate was detected, although the r e a c t i o n r a t e s were i n f l u e n c e d by pK and t h e r e was no e f f e c t from the s a l t s of seawater nor a 2+ requirement f o r t r a c e - m e t a l ions (such as Cu ). The formation of l e u c o p t e r i n as an intermediate was s p e c t r o s c o p i c a l l y and chromatograph!cally detected i n a l l cases excepting i n the pH 10 b u f f e r . I t appears t h a t i n t h i s l a t t e r case the d e t e c t i o n of l e u c o p t e r i n would not have been p o s s i b l e s i n c e l e u c o p t e r i n i t s e l f shows a f a s t e r r a t e of disappearance at pH 10.0 than the r a t e of r e a c t i o n shown by x a n t h o p t e r i n . Eased on these c o n s i d e r a t i o n s and the molecular chemistry of x a n t h o p t e r i n discussed before, a general Scheme C ( F i g . 43) i s proposed f o r the r e a c t i o n of x a n t h o p t e r i n i n seawater and at a l l p'H's s t u d i e d i n v o k i n g a 1:1 o x i d a t i v e breakdown v i a l e u c o p t e r i n . The nature of the f i n a l breakdown products i s not known and was not f u r t h e r i n v e s t i g a t e d . 50. Chelation In general, a l l the 6,7-dihydroxylated pteridines (dioxylumazine, leucopterin, 6,7-dihydroxypteridine) tested showed a s t r i k i n g comparable difference i n the i n i t i a l absorp-t i o n spectra determined i n seawater from those i n buffer of comparable pH. Since comparable differences were obtained 2+ 2 + between the buffer f o r t i f i e d with Ca (or Mg ) and synthetic 2+ 2+ 2 + seawater lacking the major divalent cations (Ca , Sr , Mg ), i t was inferred that the spectral differences were due to s p e c i f i c i n t e r a c t i o n between these pteridines and the above-mentioned divalent cations. Presumably the i n t e r a c t i o n involved complexations by chelation of the divalent cation with the Cg- and Cy-hydroxyl groups, since the above-observed s p e c t r a l differences were not observed with a l l the other pteridines tested which lacked the s t r u c t u r a l feature combining both these hydroxyl groups. Such complexation i s also supported by the chromatographic evidence (see Results), which has shown that leucopterin and dioxylumazine both show d i f f e r e n t values on i s o l a t i o n from seawater than when they were i s o l a t e d from buffered solutions. A further insight into the nature of the complexes was gained from the p a r t i c u l a r l y close s i m i l a r i t y of t h e i r spectra i n seawater to those of the uncomplexed pteridines in the buffer at pH 10.0 ( F i g . 7 ) . This s p e c t r a l s i m i l a r i t y is expected to r e f l e c t a close s t r u c t u r a l s i m i l a r i t y of the complexed pteridine species to the uncomplexed dianionic species of the 6,7-dihydroxylated pteridines known to occur at pH 10.0 ( F i g . 44-46). These considerations indicate that the divalent 51. cation complexes of these pteridines i n seawater may e n t a i l the formation of a 5-membered ring chelate of the type represented i n Figure 47A; such structures appear to be thermodynamically f e a s i b l e and would further be expected to r e s t r i c t the pteridine chromophore to an aromatic form s p e c t r a l l y i n d i s t i n g u i s h a b l e from that of the uncomplexed dianionic species of 6,7-dihydroxy-lated p t e r i d i n e s . Pteridine complexes of t h i s type (Fig. 47A) have not been previously reported, although other types have been described. Five-membered r i n g chelates involving divalent metal ion ligand formation with C^-hydroxyl and of one or two pteridine equivalents (Fig. 47E and C) have been reported by Albert (1950, 1953a, 1953b). The p o s s i b i l i t y of the production of such pter i d i n e complexes i n seawater i s not excluded, but t h e i r presence ( i f at a l l ) was not evident from the li m i t e d scope of t h i s i n v e s t i g a t i o n ; i n any case t h e o r e t i c a l considerations would appear to rule out such complexes i n the above discussed i n t e r -p r etation of the effects of seawater divalent cations on the 6,7-dihydroxylated p t e r i d i n e s . There i s , however, considerable l i k e l i h o o d of involvement of such complexes (of type reported by Albert) i n the s p e c i a l case of dark oxidation of oxylumazine 2 + requiring trace-metal ions (e.g. Cu ) schematically represented i n Figure 43A, where the Cu" -complexed species i s l e f t undefined for lack of s u f f i c i e n t information on i t s nature. The complexation of 6,7-di.hydroxylated pteridines with divalent (alkaline-earth) cations observed i n t h i s study finds i n t e r e s t i n g p a r a l l e l s i n well established complexes of benzenoid compounds with v i c i n a l hydroxyl groups. Five-membered r i n g chelates ha-^e been reported f o r c a t e c h o l and i t s d e r i v a t i v e s (Athavale et a l . , 1966, 1968; Rohrscheid et a l . , 1965; Tyson and K a r t e l l , 1968), which may i n v o l v e l i g a n d : c a t i o n s t o i c h i o -metry of 1:1 ( F i g . 47D) or 2:1 ( F i g . 47S), depending on the r e l a t i v e excess c o n c e n t r a t i o n of l i g a n d or c a t i o n present. S i m i l a r c o n s i d e r a t i o n s of the large excess of the d i v a l e n t c a t i o n s of seawater r e l a t i v e t o the d i s s o l v e d p t e r i d i n e s used i n t h i s i n v e s t i g a t i o n i n c r e a s e the p r o b a b i l i t y of 1:1 s t o i c h i o -metry of the complex of seawater c a t i o n w i t h the 6,7-dihydroxy-l a t e d p t e r i d i n e s ; hence t h i s stoichiotr:etry is. deduced (without f u r t h e r evidence) f o r the complex represented i n Figure 47A. L i g h t Reactions The molecular s u b s t i t u t i o n p a t t e r n of the pyrazine r i n g tended t o determine the r e a c t i o n r a t e s of the p t e r i d i n e s i n l i g h t , s i m i l a r t o the dark r e a c t i o n r e s u l t s i n seawater. /Jhenever s u b s t i t u e n t s were present, f i r s t - o r d e r k i n e t i c s were observed, and, when no s u b s t i t u e n t s were present, the k i n e t i c s were zero-order. This suggested t h a t 6- or 7- or both hydroxyl s u b s t i t u e n t s were re q u i r e d f o r f i r s t - o r d e r r e a c t i o n k i n e t i c s , w i t h the 6-hyd.roxylated comp6und showing the g r e a t e s t r e a c t i v i t y The a d d i t i o n of a 7-hydroxylgroup t o 6 - h y d r o x y l p t e r i d i n e s tended t o decrease t h e i r r e a c t i v i t y i n seawater; whereas the presence of a 6-hydroxylgroup had no e f f e c t on the f i r s t - o r d e r r e a c t i o n rates of 7 - h y d r o x y l p t e r i d i n e s . In the absence of pyrazine r i n g s u b s t i t u t i o n , the observed zero-order k i n e t i c s were i n f l u e n c e d by -Ntfo or -OH s u b s t i t u e n t s at Co. The hydroxylgroup, i n 53. agreement w i t h previous reported r e s u l t s ( A l b e r t , Brown, and Cheeseman, 1952b), c o n t r i b u t e d t o a g r e a t e r extent t o the s t a b i l i z a t i o n of the p t e r i d i n e nucleus than d i d the amino-group. The p h o t o l y s i s r a t e s of the p y r a z i n e - r i n g s u b s t i t u t e d p t e r i d i n e s showed s t r i k i n g e f f e c t s from pH ( i n the b u f f e r t e s t s ) , which e f f e c t s were c h a r a c t e r i s t i c of the type of s u b s t i t u t i o n . These ra t e changes occurred i n s i m i l a r ranges of pH f o r s t r u c t u r a l l y analogous p t e r i d i n e s , suggesting t h a t the pH e f f e c t was governed by the molecular species p r e v a l e n t i n these pH ranges. I o n i c or r e l a t e d s p e c i e s , dependent on the r i n g s u b s t i t u e n t s , would be expected to be determined by the pM of the medium. For each p t e r i d i n e , the molecular species expected at each pH value i s presented (as computed from p'iiCa values reported by A l b e r t (1963), and Kwietny and Bergmann (1959)) i n the f o l l o w i n g f i g u r e s : dioxylumazine, Figure 44; isoxanthop-t e r i n , F i g u r e 43; l e u c o p t e r i n , Figure 45; oxylumazine, Figure 42; and x a n t h o p t e r i n , Figure 41. When the pH-dependent changes i n molecular s t r u c t u r e are compared w i t h the pH-photolysis r a t e p r o f i l e s , d e f i n i t e patterns emerge which appear t o be charac-t e r i s t i c of s t r u c t u r a l l y analogous p t e r i d i n e s . Excepting the behaviour of i s o x a n t h o p t e r i n at pM 4.0-6.0, the i o n i z a t i o n of the 6-hydroxyl and/or the 7-hydroxjrlgroup was observed t o correspond c l o s e l y w i t h the pH range causing the most r a p i d change i on iza t ions > i n p h o t o l y s i s r a t e s . I t i s b e l i e v e d t h a t these i n f o r c i n g the p y r a z i n e r i n g t o assume an aromatic form, may render t h i s r i n g u n stable t o i l l u m i n a t i o n , i n view of the previous r e p o r t ( A l b e r t and Yamamoto, 1963b) t h a t as the number of doubly 54. bonded n i trogen atoms of p ter id ines increases a greater s e n s i t i v i t y to h y d r o l y t i c f i s s i o n i s observed. It appears, however, that t h i s r i n g i n s t a b i l i t y requires both aromat ic i ty and 6r or 7 -hydroxyl -groups, because lumazine and p t e r i n with the unsubst i tuted pyrazine r i n g i n the aromatic form ( F i g . 49) d id not show s i m i l a r r e a c t i o n k i n e t i c s (Table X I I I ) . The except ional p h o t o l y s i s - r a t e behaviour of i s © x a n t h o p -t e r i n at pH 4.0-6.0 (Fig. ' 15) i s d i f f i c u l t to exp la in and casts doubt on the p i c ture of pH-dependent d i s t r i b u t i o n of molecular species of i soxanthopter in i n Figure 48. This p i c t u r e of species d i s t r i b u t i o n was computed from data of A l b e r t and Wood • (1953) based on u n r e l i a b l e spectroscopic methods which have been known to overlook ca t ion formation i n the a c i d i c pH range (e .g . the case of hypoxanthine reported by A l b e r t and Brown, 1954). Assuming such oversight i n the case of i soxanthopter in , i t s generation of a c a t i o n i c species i n the pl-l 4 .0-6.0 range (as depicted i n F i g . 48) could o f fer a r a t i o n a l explanation of the apparently aberrant pH-dependent p h o t o l y s i s - r a t e p r o f i l e of th i s p t e r i d i n e . In th i s case, t h e o r e t i c a l considerations predic t the "lowest photo lys i s rate at the pH (around 6.0) favouring preponderance of the neutra l species and enhancement of the rates with increas ing concentrations of c a t i o n i c and an ion ic spec ies . Examples of ca t ion formation increas ing the chemical r e a c t i v i t y of p ter id ines have been reported (Inoue, and P e r r i n , 1963b) and o f f er support for thepostulated increase i n the p h o t o l y t i c a c t i v a t i o n of i soxanthopter in . The c o r r e l a t i o n of the p h o t o l y s i s - r a t e s and r e a c t i o n orders of the p t e r i d i n e s i n seawater w i t h those i n bxaffer of corresponding pH showed t h a t , w i t h the exception of dioxy-lumazine ( F i g . 1 1 ; Table X) and l e u c o p t e r i n ( F i g . 12 ; Table X I I ) they were comparably s i m i l a r i n magnitude, suggesting i n s i g n i f i c a n t e f f e c t from the s a l t s of seawater. Dioxylumazine and l e u c o p t e r i n (both 6, 7-dihydroxyl s u b s t i t u t e d ) were e x c e p t i o n a l i n showing r a p i d p h o t o l y s i s - r a t e s ( f i r s t - o r d e r k i n e t i c s ) i n seawater as opposed t o slow zero-order k i n e t i c s i n b u f f e r at the corresponding pH,. When the major d i v a l e n t -2 + 2 + 7 + metal cations (Ca , Kg , and Sr" ) were omitted from seawater (modified seawater) the r e a c t i o n k i n e t i c s were de c e l e r a t e d t o l e v e l s obtained i n the b u f f e r thereby i n d i c a t i n g t h a t these ions were r e s p o n s i b l e f o r the enhanced r e a c t i v i t y i n seawater. This e f f e c t of the d i v a l e n t - m e t a l c a t i o n s was confirmed by a 2+ 2+ study u s i n g l e u c o p t e r i n , where only Ca , Mg and KCO3 ( i o n mix medium) were i n the medium. The observed p h o t o l y s i s - r a t e and r e a c t i o n - o r d e r were i d e n t i c a l w i t h those obtained i n sea-water (Table X I I ) . The complexation of dioxylumazine and l e u c o p t e r i n w i t h the seawater d i v a l e n t - m e t a l c a t i o n s and the consequent s p e c t r o s c o p i c changes have been discussed above. I t became apparent from the s p e c t r o s c o p i c c o r r e l a t i o n s t h a t each p t e r i d i n e complex i n seawater must produce a molecular species w i t h close s t r u c t u r a l analogy t o the uncomplexed p t e r i d i n e at pH 1 0 . 0 . This s t r u c t u r a l analogy i s f u r t h e r supported by the comparable p h o t o l y s i s r e a c t i o n k i n e t i c s observed f o r these 6, 7-dihydroxylated p t e r i d i n e s i n seawater and pH 1 0 . 0 b u f f e r . T h e o r e t i c a l c o n s i d e r a t i o n s (discussed above) have ind ica ted that the enhanced aromat ic i ty of the pyrazine r i n g i n the d i a n i o n i c species may be responsible for the high r e a c t i v i t y of these p ter id ines at pl-l 10.0. A s i m i l a r c o n s o l i d a t i o n of p y r a z i n e - r i n g aromat i c i ty i s evident i n the s tructures of t h e i r complexes ( F i g . 47A) and may account for t h e i r comparably high r e a c t i v i t y . The f i n a l spectra from the prolonged photo lys i s of dioxylumazine ( F i g . 16), i soxanthopter in ( F i g . 17), l eucopter in ( F i g . 18), oxylumazine ( F i g . 19), and xanthopter in ( F i g . 20) i n seawater ind ica ted the ul t imate formation of u n i d e n t i f i e d f a r UV-absorbing, degradation products . This was a l so the case with the f i r s t - o r d e r react ions i n buffers (excepting i s o -xanthopter in at pH 4 . 0 - 5 . 0 ) . Because the degradation of the p t e r i d i n e r i n g system was expected to give r i s e to e i t h e r pyrimidine or pyrazine d e r i v a t i v e s , an attempt was made to ga in ins ight i n t o the chemical nature of the photo lys i s products by comparing t h e i r spectra with those of commercially a v a i l a b l e der iva t ives of these r i n g systems ( F i g . 50). Such comparisons i n d i c a t e d strong s i m i l a r i t y of the p h o t o l y t i c products to pyrimidine der iva t ives and suggested that the pyrazine r i n g of these p ter id ines was cleaved by the p h o t o l y s i s , presumably at the C - 7 - N 0 bond i n view of the known r e a c t i v i t y of t h i s bond to n u c l e o p h i l i c reagents (Clark , and Neath, 1966, 1963; Clark et a l . , 1969; C l a r k , and Smith, 1969). The absence of far UV-absorption from the p h o t o l y t i c product of i soxanthopter in at pll 4 .0-5 .0 indicates that t h i s reac t ion may proceed by a d i f f eren t route (probably i n v o l v i n g the c a t i o n i c species of t h i s p t e r i d i n e as discussed above). Spectroscopic monitoring of the p h o t o l y s i s of ox3'lumazine and x a n t h o p t e r i n i n seawater and i n the d i f f e r e n t b u f f e r s i n d i c a t e d the t r a n s i e n t appearance of dioxylumazine and l e u c o p t e r i n r e s p e c t i v e l y , i n a l l cases, e x c l u d i n g pH 10.0; t h i s was chromatographically confirmed. C a l c u l a t i o n s of the s t o i c h i o m e t r y of the degradatioiis of the 6-hydroxylpteri.dines v i a t h e i r 6,7-d i h y d r o x y l d e r i v a t i v e s i n d i c a t e d that the former were being degraded by an a d d i t i o n a l r e a c t i o n not i n v o l v i n g the l a t t e r . T h i s was most evident at pH 4.0, where the slow zero-order r e a c t i o n k i n e t i c s f a c i l i t a t e d c o n t r o l over the s t o i c h i o m e t r i c computations. The f a i l u r e t o detect t h e A d e r i v a t i v e s from p h o t o l y s i s i n pH 10.0 b u f f e r may be e i t h e r due t o t h e i r more r a p i d degradation than t h e i r formation from the 6-bydroxyl-p t e r i d i n e , or complete absence of such formation i n which case the l a t t e r may be degrading e n t i r e l y by the above mentioned a d d i t i o n a l r e a c t i o n not i n c l u d i n g the former. Eased on these l i n e s of experimental evidence and the t h e o r e t i c a l c o n s i d e r a t i o n s d i s c u s s e d p r e v i o u s l y (see s e c t i o n on dark r e a c t i o n s ) the f o l l o w i n g general mechanism i s proposed f o r the p h o t o l y s i s of 6-hydroxyl-p t e r i d i n e s . +H90 7,8-covalent l i g h t 6-hydroxyl- " hydrate of • >- 6, 7-dihydroxy-p t e r i d i n e ^ 6-hydroxyl- pH 4-8 p t e r i d i n e 2 Pteridine or v. . „ : J seawater pH 4.0-10.0 or seawater \sea-watc Af Light \ ater l i g h t u n i d e n t i f i e d l i g h t d i v a l e n t products < 1 c a t i o n ( p y r i m i d i n e d e r i v a t i v e s ? ) complex u n i d e n t i f i e d products ( p y r i m i d i n e d e r i v a t i v e s ? ) 58. Apart from the case of r i b o f l a v i n , which w i l l not be considered here, t h e r e have been few s t u d i e s of p t e r i d i n e p h o t o l y s i s reported i n the l i t e r a t u r e and these g e n e r a l l y r e l a t e d t o e f f e c t s of u l t r a v i o l e t i r r a d i a t i o n or s u n l i g h t w i t h r a r e attempts t o i d e n t i f y the products formed. UV- or s u n l i g h t was r e p o r t e d t o photolyse 2 , 4 , 7 - t r i h y d r o x y p t e r i d i n e and f o l i c a c i d (Shaw et a l . , 1966) and g l y o x y l i c a c i d was i d e n t i f i e d among the products obtained from the former. 6,7 - d i h y d r o x y p t e r i d i n e , 2-, 4-, and 7-hydroxypteridine were p h o t o - a l t e r e d by U V - l i g h t , and the l a s t named was found t o be reduced t o 5,6-dihydro-7-hydroxypteridine i n the presence of a hydrogen donor ( A l b e r t , 1956; A l b e r t , Frown, and Cheeseman, 1952a). Several n a t u r a l l y o c c u r r i n g p t e r i n s w i t h 3-carbon c h a i n s u b s t i t u e n t s at Cg ( b i o p t e r i n , i c h t h y o p t e r i n , s e p i a p t e r i n ) have been observed t o undergo p r o g r e s s i v e breakdown or complete cleavage of the Cg-s i d e c h a i n from exposure t o s u n l i g h t or U V - i r r a d i a t i o n (Descimon, 1971; F o r r e s t , and M i t c h e l l , 1954; Kauffmann, 1959; K u s h i b i k i et a l . , 1951; Lee et a l . , 1969; Mori et a l . , i 9 6 0 ; .^ awa, i960; Z i e g l e r , 1962), but these s t u d i e s were not extended t o f u r t h e r breakdown of the p t e r i d i n e r i n g s k e l e t o n . The present i n v e s t i g a -t i o n appears t o be the f i r s t attempt at a systematic study of the s t a b i l i t y of p t e r i n , lumasine and t h e i r 6-, 7-, 6,7-hydroxyl-ated d e r i v a t i v e s i n a q u a t i c systems, and p a r t i c u l a r l y of t h e i r a b i l i t y t o withstand exposure t o v i s i b l e l i g h t such as obtained from cool-white f l u o r e s c e n t lamps used f o r c u l t u r i n g algae and p l a n t s . In t h i s connection, i t i s pointed out that there i s no i n f o r m a t i o n i n the published l i t e r a t u r e on the e f f e c t s of any k i n d of l i g h t ( i n c l u d i n g U V - i r r a d i a t i o n ) on these p t e r i d i n e s . 59 . Purines In c o n t r a s t t o the p t e r i d i n e s ( p i - d e f i c i e n t compo-ands) where h y d r o x y l a t i o n or amination increases s t a b i l i t y , the reverse i s r e p o r t e d t o occur w i t h the purines ( p i - e x c e s s i v e compounds) ( A l b e r t , and Brown, 1954). Furthermore, i n c r e a s e i n the number of h y d r o x y l s u b s t i t u e n t s i s s a i d t o decrease the s t a b i l i t y of purines ( A l b e r t , and Brown, 1954). The r e s u l t s of the present study have shown that o n l y u r i c a c i d (a t r i - h y d r o x y l a t e d purine) was unstable i n a seawater medium i n the dark, w h i l s t the other purines (with fewer hy d r o x y l and amino s u b s t i t u e n t s ) s t u d i e d were s t a b l e . U r i c a c i d has been reporte d t o decompose at a l k a l i n e pTI ( G r i f f i t h s , 1952; S t a h l , 1969); under which c o n d i t i o n s the mono- and d i - h y d r o x y l a t e d purines were r e l a t i v e l y S t a b l e ( A l b e r t , and Brown, 1954). Apart from u r i c a c i d , the dark r e a c t i o n r e s u l t s of t h i s study agree w i t h the purine s t a b i l i t i e s r eported i n the l i t e r a t u r e and suggest that seawater media may have l i t t l e ( i f any) e f f e c t on purine s t a b i l i t y . U r i c a c i d and xanthine showed s i g n i f i c a n t p h o t o l y t i c decomposition i n the seawater medium of t h i s study, w h i l s t the other purines t e s t e d were h a r d l y a f f e c t e d by the i l l u m i n a t i o n source. There have been s e v e r a l s t u d i e s on the p h o t o l y s i s of purines reported i n the l i t e r a t u r e , but these g e n e r a l l y r e l a t e t o the e f f e c t s of u l t r a v i o l e t i r r a d i a t i o n . Adenine, guanine, hypoxanthine, xanthine and u r i c a c i d were reported t o undergo UV - p h o t o l y t i c decomposition, but few attempts have been made t o i d e n t i f y the decomposition products ( C a n z a n e l l i et a l . , 1951; F e l l i g , 1954; Kland, and Johnson, 1956; Loofbourow, and Stimson, 1940). Purine r e a c t i v i t y t o U V - i r r a d i a t i o n i s reported t o be 60. dependent on the number of carbonyl groups i n the r i n g system w i t h a g r e a t e r c o n t r i b u t i o n from the carbonyl group at C2 (Loofbourow, and Stimson, 1940; C a n z a n e l l i et a l . , 1951). C a n z a n e l l i et aj,. (1951) l i s t the f o l l o w i n g order of decreasing p u r i n e s t a b i l i t y : adenine, hypoxanthine and guanine, xanthine, u r i c a c i d . The r e s u l t s of the present i n v e s t i g a t i o n agree w e l l w i t h t h i s scheme i n that adenine, hypoxanthine, and guanine were s t a b l e t o l i g h t w h i l e xanthine and u r i c a c i d decomposed i n i n c r e a s i n g order. Whereas decomposition i s s p e c t r o s c o p i c a l l y i n d i c a t e d by a p r o g r e s s i v e decrease i n a b s o r p t i o n i n t e n s i t y at the c h a r a c t e r i s t i c X._„,r of a p u r i n e , an i n e x p l i c a b l e p r o g r e s s i v e i n c r e a s e i n a b s o r p t i o n i n t e n s i t y was observed w i t h xanthine i n the dark, and w i t h adenine, guanine, and hypoxanthine both i n the l i g h t and dark. These increases may r e f l e c t the formation of purine molecular species of unknown type. S i m i l a r a b s o r p t i o n increases have been reported f o r adenine and xanthine i n the l i g h t ( C a n z a n e l l i et a l . , 1951; C h r i s t e n s e n , and Giese, 1954), but t h e i r causes are unknown. I t has been speculated that such a b s o r p t i o n increases may be due t o changes i n c o n f i g u r a t i o n or molecular rearrangements (Garay, and Cuba, 1954; Loofbourow, 1948). The d i f f e r e n c e i n the a b s o r p t i o n i n c r e a s e s observed i n t h i s study between the l i g h t and dark r e a c t i o n s of adenine, guanine, and hypoxanthine suggest t h a t l i g h t may a c t u a l l y have a r e t a r d i n g e f f e c t on the i n c r e a s e that would have occurred i n i t s absence. I t i s probable t h a t the components of the seawater medium may p l a y a r o l e i n t h i s phenomenon, which has not been f u r t h e r i n v e s t i g a t e d . 61. S i g n i f i c a n c e of the Light Source Used The p h o t o l y s i s studies reported i n t h i s i n v e s t i g a t i o n stemmed from a c c i d e n t a l observations of the bleaching of xa n t h o p t e r i n ( d i s s o l v e d i n a seawater medium) by l i g h t from cool-white f l u o r e s c e n t lamps (normally used f o r c u l t u r i n g algae) during t e s t s designed t o explore the p o s s i b i l i t y of p t e r i d i n e s s e r v i n g as N-source f o r growth of a marine phyto-p l a n k t e r . Whereas previous reports of the p h o t o l a b i l i t y of c e r t a i n p t e r i d i n e s had been s o l e l y concerned w i t h e f f e c t s of U V - i r r a d i a t i o n , t h i s b l e a c h i n g of x a n t h o p t e r i n from a v i s i b l e l i g h t source was t o t a l l y unexpected and c a l l e d f o r f u r t h e r s t u d i e s w i t h t h i s l i g h t source, e s p e c i a l l y s i n c e such p h o t o l y t i c e f f e c t s would negate the v a l i d i t y of these and r e l a t e d compounds s e r v i n g d i r e c t l y as M-source f o r growth of micro-algae. This doubt was indeed j u s t i f i e d by the p h o t o l y s i s r e s t i l t s r e p o r t e d above, which lead t o the c o r o l l a r y t h a t the s t a b i l i t y of a compound must be e s t a b l i s h e d under the c u l t u r e c o n d i t i o n s used before i t may be assumed t o support m i c r o b i a l or a l g a l growth, f o r any growth observed may be a c t u a l l y due t o a c h e m i c a l l y m o d i f i e d or degraded product of the compound. Apart from such c u l t u r e c o n s i d e r a t i o n s , the l i g h t source used i s a l s o r e l e v a n t t o p h o t o l y s i s of organic compounds from s u n l i g h t in oceanic waters. Suggestions of o x i d a t i v e or p h o t o l y t i c degradation of d i s s o l v e d organic matter i n the oceans, at l e a s t i n the euphotic zone, have been made by Harvey (1955) and Home (1969). Measurements of the r a d i a n t energy and s p e c t r a l d i s t r i b u t i o n from the l i g h t source used i n the present study i n d i c a t e that ( a f t e r allowance i s made f o r a b s o r p t i o n of the near U V - l i g h t 62. by the glass of the t e s t - f l a s k s ) both are comparable to solar r a d i a t i o n normally penetrating oceanic water, at least i n the UV- and v i s i b l e regions; the greater i n t e n s i t y of red and i n f r a -red r a d i a t i o n from sunlight are reported to be r a p i d l y absorbed at the water surface (Poole et a l . , 1937; K o l l e r , 1965; Strickland 1958). It thus appears that the photolytic degradations of purines and pteridines observed i n t h i s stud}? may be v a l i d l y extended to predict s i m i l a r fate of these compounds i n the marine environment. To what extent.the environment temperature w i l l a f f e c t these degradations i s unknown. B i o l o g i c a l and Ecological Significance of the Results Pteridines The b i o l o g i c a l s i g n i f i c a n c e of the results of this i n v e s t i g a t i o n may be p a r t i c u l a r l y relevant to the ecological turnover of the pteridines studied i n the aquatic environment. i P t e r i n , xanthopterin, isoxanthopterin, and leucopterin have been found i n s i g n i f i c a n t amounts i n ce r t a i n b i o l o g i c a l systems ( b u t t e r f l y wings, amphibian, f i s h , and r e p t i l e skin) and, although t h e i r exact function i s not known, they are at present believed to be products of catabolism of known "biochemically-involved" pteridines (such as f o l i c acid, b i o p t e r i n , etc.) (Rembold, 1970; Rembold et a l . , 1969, 1971). Furthermore, t h e i r deaminated counterparts (lumazine, oxylumazine, and dioxylumazine) have been i d e n t i f i e d i n pathways of t h e i r corresponding microbiological breakdown (e.g. McNntt, 1962: Levy and McNutt, 1962; Levenbergand, and Hayaishi, 1959; 63. Rembold, and Simmersbach, 196?). In general, the e x i s t i n g l i t e r a t u r e has v i r t u a l l y ignored the catabolic degradation of the ri n g system of these pteridines and the mechanisms involved. Since there has been no geochemical evidence for the accumula-t i o n of refractor}' pteridines i n nature, i t may be inf e r r e d that they undergo appreciable turnover e i t h e r through b i o l o g i c a l or non-biological agencies. Such turnover may be expected to be more re a d i l y i d e n t i f i a b l e i n aquatic systems i n view of the d i s p e r s i b i l i t y of the pteridines effected by such systems. Viewed i n th i s e c o l o g i c a l context, the present i n v e s t i g a t i o n indicates that the non-biological components of the aquatic environment (such as s a l i n i t y , pH, l i g h t , and trace-metal ions) can cause appreciable chemical degradation of xanthopterin, isoxanthopterin, oxylumazine, leucopterin, and dioxylumazine; a l b e i t such degradation may be slow i n certain cases but i t i s considered s u f f i c i e n t to preclude t h e i r constant accumulation and to maintain s i g n i f i c a n t turnover. Purines The widespread occurrence of c e r t a i n purines (adenine, guanine) as components of nucleic acids i n a l l forms of l i f e i s w e l l known and t h e i r pathways of endogenous catabolic degradation or exogenous microbial breakdown v i a t h e i r deaminated counterparts (hypoxanthine, xanthine) are well established (e.g. White et a_l. , 1968). However, apart from t h e i r appearance i n chemically combined form i n genetic and related biochemicals, they are also known to be produced as free bases (e.g. u r i c acid, guanine, hypoxanthine) i n considerable amounts i n f i s h s k i n and i n excrements of animals and b i r d s . The 64. l i t e r a t u r e i s replete with reports of microbial u t i l i z a t i o n of free purines as N-source f o r growth and the microorganisms studied include b a c t e r i , fungi, and u n i c e l l u l a r algae. It appears, therefore, that b i o l o g i c a l agencies play an important rol e i n the turnover of purines i n the aquatic and t e r r e s t r i a l environment. The r o l e of chemical (non-biological) agencies i n t h i s turnover has not been previously assessed. The l i t e r a t u r e reports on chemical degradation of purines have been p r i m a r i l y concerned with effects from o'-rays, X-rays or far U V - i r r a d i a t i o n . The resul t s of the present i n v e s t i g a t i o n indicate that some purines (adenine, guanine, hypoxanthine) may be expected to be quite stable and others (uric a c i d , xanthine) to be unstable i n the oceanic environment. The i n s t a b i l i t y of xanthine appears to be subject to the i n t e n s i t y of sunlight a v a i l a b l e , while u r i c acid would be unstable under a l l conditions excepting unknown effects of temperature not examined here. In view of the a v a i l a b l e geochemical evidence i n d i c a t i n g no substantial accumulation of purine deposits (with few exceptions^") i n the oceanic encironment, i t may be inferred that the chemically stable purines are degraded s o l e l y by b i o l o g i c a l agencies and that the chemically unstable ones are degraded by both b i o l o g i c a l and chemical agencies. The p o s s i b i l i t y arises that u r i c acid may tend to acciamulate from p r e c i p i t a t i o n out of seav/ater owing to i t s d i f f i c u l t s o l u b i l i t y in- seawater. This property of u r i c a c i d , i n fact , created problems i n the present in v e s t i g a t i o n such as guano deposits from b i r d droppings on rocky shores and i s lands. 65. and a clear—cut answer to the question of i t s s t a b i l i t y i n sea-water could only be obtained with very low concentrations insuring i t s complete d i s s o l u t i o n . A s i m i l a r problem was encountered by Birdsey (1962) studying the u t i l i z a t i o n of u r i c acid as PJ-source for growth of marine phytoplankters but was erroneously interpreted by t h i s author leading to the inference that u r i c acid was stable i n seawater. The chemical i n s t a b i l i t y of u r i c acid observed i n the present study offers s u f f i c i e n t basis to argue against i t s possible accumulation, even i n insoluble form, i n the marine environment. The insoluble u r i c a c i d would be expected to be i n continous equilibrium with i t s soluble form, and the l a t t e r would be degraded both chemically and b i o l o g i c a l l y . 66. TABLE I. S o l u b i l i t y of pteridines and purines i n d i s t i l l e d water at 20°C, COMPOUND: Maximuny.v Cone, used (mM) Cone. (mM)^ i n misc. media i n present study. P t e r i d i n e s : Lumazine 7.62 0.04 6,7-dihydroxypteridine 2.03 0.04 Oxylumazine 0.75 0.04-0.045 Xanthopterin 0.14 0.04-0.05 Pt e r i n 0.108 0.04 Dioxylumazine 0.088 0.04 A . ( 2 ) Isoxanthopterin 0.028 0.03^2^ Leucopterin 0.007 0.02' Purines: Adenine 6.7 0.04 Hypoxanthine 5.2 0.04 Xanthine 3.3 0.054 Guanine 0.033 0.02 Uric Acid 0.15 0.05, 0.022, and 0.007 (1) calculated from data presented by P f l e i d e r e r (1963). (2) saturated solutions but no p r e c i p i t a t i o n occxirred. TABLE I I . Summary of molar e x t i n c t i o n c o e f f i c i e n t s (€ x 10" ) of pteridines at t h e i r c h a r a c t e r i s t i c absorption maxima (X) i n the 295-400 nm. range. MEDIA: SEAWATER ION BUFFER SOLUT PTERIDINE PAPA SYNT. MODF. MIX 10.0 9.0 8.0 7.0(D 6.0-Dioxylumazine € 13.1 13.1 12.1 11.1 12.0 12.3 12.4 12.0 X 338 338 345 346 345 345 345 344 £ mm 11.9 10.9 11.6 12.2 12.4 12.0 X 330 336 333 331 330 330 Isoxanthopterin € 12.2 11.9 mm mm 11.8 11.8 11.9 12.1 12.6 X 334 335 338 334 333 338 341 € — MP mm mm 12.0<4> 12.8 X 338 340 Leucopterin € 9.38 9.44 8.67 9.41 8.70 8.76 8.50 7.12<5> 7.30 X 339 339 345 338 345 345 344 341 335 € 8.44 *• 8.63 8.55 8.50 9.36 12.3 X 335 337 334 335 298 298 Lumazine € 4.31 4.27 mm 4.47 4.26 X 335 335 347 335 Oxylumazine € 6.83 6.75 6.75 _» 6.18 mm 6.84 6.69 5.54 X 380 382 381 393 382 380 375 5.0<2> (5.4) (5.4) 4.0 1.0 (3) (5.0) (5.0) (5.0) 9.86 8.43(8.43 a) 343 327 (327) 10.7 -332 13.0 12.0(10.8 b) 341 341 (340) 6.35 6.56 -338 337 11.1 10.7 -299 299 - 5.58 -325 5.22 5.17 -366 366 TABLE II (contd.) MEDIA: PTERIDINE SEAWATER PAPA SYNT. MODF. ION MIX 10.0 9.0 8.0 BUFFER 7.0(D SOLUTIONS (pH) 6.0-5.0 ( 2 ) 4.0 1.0<3> Pt e r i n € X 5.90 360 6.03 360 - - 6.58 359 - 5 . 5 9 360 - - - 7.06(7.0 C) 313 (312) Xanthopterin € X 5.74 392 5.61 393 5.55 390 5.69 393 6.22 394 - 5.51 392 4.43 390 2.50 385 4.55(4.50 d) 356 (356) 6,7-dihydroxypteridine € X 13.8 331 13.8 333 13.7 334 - 15.0 335 12.8 335 13.2 335 9.86 334 3028<6-°> 14.3 300 14.2 305 € X 18.4 318 18.1 318 20.6 318 - 19.2 322 20.0 20.0 319 319 16.2 318 - - -e X - - 15.9 308 - - 15.5 310 15.7 309 14.9 309 - -(1) phosphate buffer. (2) pH i n brackets. (3) values i n parentheses reported i n l i t e r a t u r e : a) Levy and McNutt (1962); b) V i s c o n t i n i et a l . (1955); c) Stokstad et a l . (1948); d) Stuart et a l . (1964). (4) T r i s b u f f e r . (5) corresponding values for leucopterin i n T r i s b u f f e r are £ = 7.60 at X = 340 and € = 8.59 at X = 298. ON '00 TABLE I I I . Summary of molar e x t i n c t i o n c o e f f i c i e n t s (£ x 10~ 3) of purines at t h e i r c h a r a c t e r i s t i c absorption maxima a ) i n the 225-•300 nm. range. N S M ^ MEDIUM BUFFER SOLUTIONS^3) LITERATURE VALUES PURINE: pH X e pH X pH X £ Adenine Ca. 1.0<2> 263 11.7 1.0 263 11.7 1-3 262.5 13.15 7.6 260 11.9 8.0 260 11.9 6-8 260.5 13.35 (a) Guanine Ca. Ca. 1.0 1.0 248 273 15.1 7.83 1.0 1.0 247 274 10.1 7.44 1-2 1-2 248.5 275.5 11.4 7.35 (a) 7.6 7.6 246 276 11.5 7.54 8.0 8.0 246 276 9.60 8.07 7 7 246 275.5 10.7 8.15 (a) Hypoxanthine Ca. 1.0 249 13.2 1.0 249 9.94 1.0 248 10.9 7.6 249 12.8 8.0 250 9.96 4 r 7 249.5 10.7 (a) U r i c acid Ca. Ca. 1.0 1.0 230 285 9.81 12.3 1.0 1.0 230 285 7.95 11.1 2 2 231 283 8.5 11.5 (b) 7.6 7.6 236 292 10.1 12.6 8.0 8.0 236 292 7.65 9.96 9 9 235 292 9.6 11.6 (b) Xanthine Ca. Ca. 1.0 1.0 227 262 7.14 10.4 1.0 1.0 228 262 6.20 8.84 0 0 231 260 6.35 9.15 (c) 7.6 7.6 243 272 7.77 9.82 8.0 8.0 244 274 7.26 8.35 2-6 10 10 267 240.5 277.5 10.25 8.9 9.3 (a) (a) TABLE III (contd.) Footnotes: (1) see Appendix B. (2) obtained by add i t i o n of one drop ION HC1 to the cuvette containing 1 ml soluti o n of the purine i n NSM-medium at pH 7.6. (3) purine solutions i n HC1-KC1 buffer (pH 1) or sodium phosphate b u f f e r (pH 8). References: (a) Dawson et a l . (1969). (b) C a v a l i e r i et a l . (1950). (c) Sober (1968J. TABLE IV. Dark reaction r a t e s o f pteridines i n various media at room temperature. PTERIDINE PAPA SEAWATER SYNT. MODF. ION MIX 10.0 9.0 BUFFER 8.3 SOLUTIONS 8.0 7.0 (pH) 6. 0-5.0 4.0 Dioxylumazine 0.13 N.D.<2> 0.19 - 0.60 0.25 0.13 N.D. N.D. - N.D. Is oxant hopt e r i n N.D. N.D. - - N.D. N.D. N.D. N.D. N.D. N.D. N.D. Leucopterin 0.20 0.60 0.13 0.34 0.84 0.42 0.28 0.21 0.12 - 0.10 Lumazine N.D. N.D. - - N.D. - - N.D. - - -Oxylumazine 5.40 ( 3 ) I O . O ^ 3 ) 8.90<3) - N.D. - - N.D. N.D. N.D. N.D. Pt e r i n N.D. N.D. - - N.D. - - N.D. - - -Xanthopterin 0.62 1.10 0.48 0.32 0.61 - - 0.46 0.40 N.D. 0.48 (1) zero order reaction rates expressed as jomoles pter i d i n e modified per l i t e r per 100 hours. (2) N.D. - not detectable. (3) roughly translated from f i r s t - o r d e r reaction k i n e t i c s (see text) to zero-order reaction rates. TABLE V. The Rf values of oxylumazine dark oxidation products and authentic compounds on paper chromatograms. Compound: propan-l-ol:water: NH/.OH (15N) R f VALUES IN: aqueous NH4C1 (3%) buta n - l - o l : a c e t i c acid:water Oz : 5": 3 ) Synthetic seawater media -authentic oxylumazine -oxylumazine oxidation product -authentic dioxylumazine -authentic dioxylumazine and oxylumazine oxidation product Sodium chloride (0.8M) media -authentic oxylumazine -oxylumazine oxidation product -authentic dioxylumazine -authentic dioxylumazine and oxylumazine oxidation product 0.18 0.07 0.06 0.06 0.19 0.13 0.14 0.14 0.47 0.36 0.36 0.36 0.47 0.34 0.34 0.34 0.24 0.04 0.04 0.04 0.24 0.03 0.03 0.03 -^1 ro 73 TABLE VI. F i r s t order reaction rates and reaction stoichiometry for the oxylumazine dark oxidation i n various media. MEDIA: Reaction rates K x 10 3 (hours - 1) St Reaction . oichiometry'^ ' PAPA seawater 1.47 2:1 Synthetic seawater 1.98 2:1 Modified seawater 1.89 2:1 Various NaCl solutions see Fig 3 a l l 1:1 0.5 M NaCl^ 3) 1.05 1:1 plus N a H C 0 3 ^ 4 ^ 1.17 1:1 plus Na 2 S 0 4 0.99 1:1 plus H3BO3 1.58 1:1 plus H3BO3 and Na2SO^ 0.59 1:1 plus NaHCOg and H3BO3 0.73 1:1 plus N a H C 0 3 and Na2S0^ 0.90 1:1 plus NaHCOg, Na2SC>4 and H3BO3 0.64 1:1 plus KBr 0.46 1:1 plus KBr and NaF 0.57 1:1 0.5 M KCl<3> 1.04 1:1 (1) for disappearance of oxylumazine. (2) r a t i o of oxylumazine to dioxylumazine. (3) buffered at pH 8.0. (4) concentrations of a l l additives shown were the same as used i n preparing the synthetic seawater. 74. TABLE VII. 9 + 2 + Fe" and Cu analysis of f i l t e r e d media used for oxylumazine dark oxidation s t u d i e s . MEDIA: Fe + Cu pM. pH PAPA seawater 0.01 0.18 Synthetic seawater 0.03 0.13 Modified seawater 0.15 0.65 0.5 M NaCl^ 2) 0.59 0.22 ca. 0.58 ( 3 ) (1) analysis by the method of Strickland and Parsons (1968) on membrane f i l t e r e d media samples. (2) buffered at pH 8.0 with 0.05 M sodium phosphate. (3) calculated from impurities listed on reagent bottles. 75. TABLE V I I I . 7 + 2 + The e f f e c t o f Fe and Cu on t h e o x y l u m a z i n e d a r k o x i d a t i o n r e a c t i o n i n v a r i o u s media. MEDIA: F i r s t o r d e r r e a c t i o n r a t e R e a c t i o n K x 1 0 3 ( h o u r s " 1 ) S t o i c h i o m e t r y 0 . 0 5 M phosphate b u f f e r pH 8 . 0 no r e a c t i o n -p l u s F e 2 + ( 0 . 5 7 p M ) ( 2 ) no r e a c t i o n -p l u s F e 2 + ( 0 . 5 7 pK)^l} and C u Z + ( 0 . 1 9 ; J M ) U ; no r e a c t i o n -0 . 5 M N a C l ( 3 ^ 1 . 2 2 1 : 1 p l u s E D T A ( 5 . 0 ;uM) no r e a c t i o n -p l u s E D T A ( 5 . 0 uM) and F e 2 + ( 5 . 0 pM) no r e a c t i o n -p l u s E D T A ( 5 . 0 ; J M ) and C u 2 + ( 5 . 0 pM) 0 . 7 5 1 : 1 S y n t h e t i c s e a w a t e r 1 . 7 1 2 : 1 p l u s E D T A ( 5 . 0 pH) no r e a c t i o n -p l u s E D T A ( 5 . 0 pK) and F e 2 + ( 5 . 0 ; J M ) no r e a c t i o n _ p l u s E D T A ( 5 . 0 pK) and C u 2 + ( 5 . 0 pK) 1 . 9 5 2 : 1 ( 1 ) r a t i o o f o x y l u m a z i n e t o d i o x y l u m a z i n e . ( 2 ) t h e s e c o n c e n t r a t i o n s were c a l c u l a t e d t o r e p r e s e n t t h e known t r a c e m e t a l i m p u r i t i e s i n 0 . 5 M N a C l . ( 3 ) b u f f e r e d a t pH 8 . 0 w i t h 0 . 0 5 M sodium p h o s p h a t e . TABLE IX. Ki n e t i c evidence f o r the formation of leucopterin from the dark re a c t i o n of xanthopterin i n various media. Reaction rates f o r the disappearance of xanthopterin and f o r the appearance of leucopterin are presented. The difference between these rates i s compared with the expected simultaneous leucopterin disappearance rate. Simultaneous leucopterin disappearance rate MEDIA: Xanthopterin Leucopterin Calculated from Values from disappearance appearance xanthopterin authentic^} leucopterin*' ' rate rate disappearance rate minus leucopterin appearance rate PAPA seawater 0.62 0.37 0.25 0.20 Synthetic seawater 1.10 0.52 0.59 0.60 Modified seawater 0.48 0.32 0.16 0.13 Ion mix 0.32 0.32 0.34 Buffered solutions (pH) 10.0 0.61 N.M. 0.61 0.84 8.0 0.46 0.32 0.15 0.21 7.0 (phosphate) 0.40 0.32 0.08 0.12 5.0 N.M. - - -4.0 0.48 0.34 0.14 0.10 (1) a l l zero order rates expressed i n uMoles pteridine modified per l i t e r per 100 hours. (2) from Table IV. (3) N.M. - not measurable. TABLE X. Photolytic decomposition rates of dioxylumazine i n various media at room temperature under cool white fluorescent lamps. MEDIA: Reaction order F i r s t K x 10 3 (hours"1) order reaction K U ) (uM/100 hours) Zero order reaction K (uM/100 hours) PAPA seawater f i r s t 1.47 6.2 -Synthetic seawater f i r s t 1.38 6.1 -Modified seawater zero - - 0.41 Buffered s o l u t i o n (pH) 10.0 f i r s t 0.56 3.1 -9.0 zero - 1.32 8.3 zero - - 0.40 8.0 zero - - 0.39 7.0 (phosphate) zero - - 0.27 4.0 zero 0.14 (1) roughly translated from f i r s t - o r d e r reaction k i n e t i c s (see text) to zero-order reaction rates, for dire c t comparison with the other reaction rates. TABLE XI. Photolytic decomposition rates of isoxanthopterin i n various media at room temperature under cool white fluorescent lamps. MEDIA: Reaction order F i r s t K x 103 (hours ) order reaction X U) (uM/100 hours) Zero order reaction K (uM/100 hours) PAPA seawater f i r s t 3.41 14.0 -Synthetic seawater f i r s t 2.77 10.9 -Buffered solutions (pH) 10.0 f i r s t 6.24 23.0 -9.0 f i r s t 3.54 16.0 -8.3 f i r s t 4.34 18.0 _ 8.0 f i r s t 3.08 12.2 -7.0 (phosphate) zero - - 4.00 7.0 ( T r i s ) zero - - 2.08 6.0 zero - - 1.90 5.0 f i r s t 3.04 12.0 _ 4.0 f i r s t 19.2 32.0 — (1) roughly translated from f i r s t - o r d e r reaction k i n e t i c s (see text) to zero-order reaction rates, f o r direct comparison with the other reaction rates. TABLE XII. Photolytic decomposition rates of leucopterin i n various media at room temperature under cool white fluorescent lamps. MEDIA: Reaction order F i r s t K x 10 3 (hours - 1) order r e a c ^ o n (uM/100 hours) Zero order reaction K (uM/100 hours) PAPA seawater f i r s t 2.46 9.8 -Synthetic seawater f i r s t 1.63 7.0 -Modified seawater zero - - 0.14 Ion mix f i r s t 1.80 7.5 -Buffered solutions (pH) 10.0 f i r s t 1.31 6.0 -9.0 zero - - 1.59 8.3 zero _ - 0.80 8.0 zero - _ 0.26 7.0 (phosphate) zero - - 0.12 7.0 ( T r i s ) zero - - 0.37 4.0 zero — - 0.10 (1) roughly translated from f i r s t - o r d e r reaction k i n e t i c s (see text) to zero-order reaction rates, f o r direct comparison with the other reaction rates. TABLE XIII. Photolytic decomposition rates of lumazine and p t e r i n i n various media at room temperature under cool white fluorescent lamps. MEDIA: Reaction Zero order reaction order K (pM/100 hours) Lumazine:-PAPA seawater Synthetic seawater Buffered solution pH 8.0 Buffered s o l u t i o n pH 10.0 P t e r i n : -PAPA seawater Synthetic seawater Buffered sol u t i o n pH 8.0 Buffered sol u t i o n pH 10.0 zero zero zero zero zero zero zero zero 0.14 0.13 0.12 0.29 0.43 0.50 0.52 0.80 TABLE XIV. Photolytic decomposition rates of oxylumazine i n various media at room temperature under cool white fluorescent lamps. Reaction F i r s t order reaction Zero order reaction Appearance of MEDIA: order K x l O 3 K^ 1) K dioxylumazine( 2). (hours-1) (uM/100 hours) (uM/100 hours) PAPA seawater f i r s t 15.7 61.0 - yes ( f i g . 24) Synthetic seawater f i r s t 16.9 68.0 - yes ( f i g . 24) Modified seawater f i r s t 17.8 73.0 - yes ( f i g . 24) Buffered solutions (pH) 10.0 f i r s t 30.1 125.0 - N.D. C 3 ) ( f i g . 25) 8.0 f i r s t 18.2 76.0 - yes ( f i g . 25) 7.0 (phosphate) f i r s t 12.2 50.0 - yes ( f i g . 26) 5.4 f i r s t 4.03 16.8 - yes ( f i g . 27) 4.0 zero — 2.20 yes ( f i g . 26) (1) roughly translated from f i r s t -order reaction k i n e t i c s (see text) to zero-order reaction rates, f o r d i r e c t comparison with the other reaction rates. (2) see figure i n parentheses for graphs of the concentration of oxylumazine and dioxylumazine versus time. (3) N.D. - not detectable. TABLE XV. Photolytic decomposition rates of xanthopterin i n various media at room temperature under cool white fluorescent lamps. MEDIA: Reaction order F i r s t K x 10 J (hours"1) order reaction KU) (uM/100 hours) Zero order reaction Appearance of. K leucopterin.' ' (uM/100 hours) PAPA seawater f i r s t 12.6 47.0 yes ( f i g . 30) Synthetic seawater f i r s t 8.34 34.0 yes ( f i g . 30) Modified seawater f i r s t 6.41 25.0 yes ( f i g . 31) Ion mix f i r s t 10.7 39.0 yes ( f i g . 31) Buffered solutions (pH) 10.0 f i r s t 11.9 41.0 N.D. ( 3 ) ( f i g . 32) 8.0 f i r s t 10.7 39.0 yes ( f i g . 32) 7.0 f i r s t 4.07 16.0 yes ( f i g . 33) 5.0 second - yes ( f i g . 34) 4.0 zero - - 5.6 yes ( f i g . 33) (1) roughly translated from f i r s t -order reaction k i n e t i c s (see text) to zero-order reaction rates. (2) see figure i n parentheses for graphs of the concentration of xanthopterin and leucopterin versus time. (3) N.D. - not detectable. (4) roughly translated from second-order reaction k i n e t i c s to a zero-order reaction r a t e . TABLE XVI. Dark reaction rates of purines i n NSM-medium^1) at room temperature. PURINE: Type of spectroscopic change(2J Time period of change (hours) Order of the reaction Rate constant (K) See f i g u r e Adenine increase increase 0-200 200-700 zero N.C. 0.09 uM/100 h o u r s ^ 36 Guanine increase increase 0-250 200-700 zero zero 1.21 ;iM/l00 h o u r s ^ ) 0.07 juM/100 hours( 4) 36 Hypoxanthine increase no change 0-200 250-700 N.C. no change N.C. 36 U r i c acid decrease 0-500 zero 0.76 pM/100 hours 37 Xanthine increase no change 0-250 250-700 N.C. no change N.C. 36 (1) see Appendix 3. (2) expressed as increase or decrease of the absorbance at the c h a r a c t e r i s t i c absorption maximum of the purine. (3) N.C. - not calculable. I n s u f f i c i e n t readings were taken during time period of change. (4) calculated on the assumption that the increase i s based on the production of an equivalent to the corresponding purine. TABLE XVII. Photolytic reaction rates of purines i n NSM-medium^1) at room temperature under cool white fluorescent lamps. PURINE: Type of spectroscopic change** ' Time period of change (hours) Order of the reaction Rate constant (K) See figure Adenine increase increase 0-50 50-700 N.C.<3> zero N.C. , 0.08 uM/100 hours 36 Guanine increase 0-700 zero 0.17 uM/100 h o u r s ^ 36 Hypoxanthine decrease increase 0-50 50-700 N.C. zero N.C. , . 0.13 uM/100 h o u r s ^ 36 Uric a c id decrease 0-700 f i r s t 3.15 x 10" 3 ( h o u r s - i ) ^ ) 37 Xanthine decrease 0-700 zero 0.34 uM/100 hours 36 (1) see Appendix B. (2) expressed as increase or decrease of the absorbance at the c h a r a c t e r i s t i c absorption maximum of the purine. (3) N.C. - not calculable. I n s u f f i c i e n t readings were taken during time period of change. (4) calculated on the assumption that the increase i s based on the production of an equivalent to the corresponding purine. (5) or 12.0 Limoles/lOO hours when roughly translated from f i r s t - o r d e r reaction k i n e t i c s (see text) to a zero-order reaction rate. 85. Figure 1. Dark oxidation of oxylumazine in synthetic seawater. I. Temporal changes ( ) of absorbances at the pH and corresponding Xmax indicated. II. Computed corresponding changes ( ) in pteridine concentration showing the disappearance of oxylumazine QOLU] and the appearance of dioxylumazine [DIOLU]. 0 200 400 600 800 1000 1200 1400 1600 T I M E (HOURS) R 6 . Figure 2 . Changes i n the absorption spectrum of oxylumazine i n synthetic seawater with the indicated periods of storage i n darkness at room temperature under aseptic conditions. 87. Figure 3. The ef f e c t of sodium chloride concentration i n 0.05M sodium phosphate buff e r (pH 7.8-7.9) on the dark oxidation rate of oxy-lumazine. (• •) NaCl alone. (o o) NaCl V 7 i t h added EDTA (5.0 uM). 2 + ( O) NaCl omitted, but Fe (0.57M) 2 + and Cu (0.19M) included. [NaCl] (moles/litei) Q P Figure 4. Changes i n the absorption spectrum of oxylumazine i n 1.0M sodium chloride (buffered at pH 7.9) with the indicated periods of storage i n darkness at room temperature under aseptic conditions. Absorption spectra of dioxylumazine and oxylumazine i n pH 8.0 phosphate buffer and a f t e r a c i d i f i c a t i o n to pH 1.0. dioxylumazine pH 8.0 ( ) pH 1.0 ( •) oxylumazine pH 8.0 ( ) pH 1.0 ( ) 90 . Figure 6. Dark oxidation of oxylumazine i n 1.0M NaCl solution buffered at pH 7.9. I. Temporal changes ( ) of absorbances of oxylumazine at the pH and corresponding Xmax indicated. I I . Computed corresponding changes ( ) i n pteridine concentration showing the disappearance of oxylumazine [puS] and the appearance of dioxylumazine []DIOLU]. T I M E (HOURS) 91. Figure 7. Changes i n the major absorption peaks of dioxylumazine ( ), leucopterin ( ), and 6 , 7-dihydroxypteridine ( ) i n various media by divalent 2+ 2 + metal ions. The Ca and Mg ion concentrations correspond to those found i n seawater. 1 1 ' 1 1 1 1 1 1 1 • 1 1 1 11 300 330 360 Buffer pH 10.0 PAPA Seawater A B Tris Buffet 5 pH8.3 + M g 2 + 0 R B /\ Tris Buffer N + C a 2 + > C E Tris Buffer pH 8.3 Modified Seawater ' I ' I' I ' I ' I 1 I ' I ' I 3 0 0 330 360 l \ I \ I \ 7 V y \ \ / \ / \ J n \ / \ / \ / \ y \ / \ ! \ \ \ \ \ i 1 1 1 1 • i 1 1 11 ' i" 300 3 3 0 / yj(pH8.j) (pH 8.5 (PH 8.1) 'I 1 l 1 I 1 I 1 I 1 I ' 1 1 I 300 330 360 nm. / / \ \ • I 1 11 I 1 1 1 I • I 1 I 1 I 300 330 360 nm. "T-t-r . . , 1 i 1 1 1 3 0 0 330 nm. Absorptioii spectra of 6 , 7-dihydroxy-pteridine i n pH 8.0 phosphate buffer, i n PAPA seawater and a f t e r a c i d i f i c a t i o n to pH 1.0. 93. Figure 9. Emission spectrum of cool-white fluorescent lamps used f o r the photolysis experiments. A - 650-700nm portion of lamp spectrum. B - ten-fold magnification of the 300-350nm spectral region. C - the ef f e c t of "Kimax" brand glass of the photolysis flasks on the spectral region of insert E. Figure 1 0 . Absorption spectrum of "Kimax" brand glass from the photolysis f l a s k s . - i 1 1 1 — — i 1 r - — i r — r — — I r 200 300 400 500 600 700 95. Figure 1 1 . The ef f e c t of pH on the photolytic reaction rate of dioxylumazine. a. represents PAFA seawater. b. represents modified seawater. 6.0 9 6 . Figure 12. The effect of pH on the ph o t o l y t i c reaction rate of leucopterin. a. represents PAPA seawater. b. represents modified seawater. igure 13. The effect of pH on the photolytic reaction rate of oxylumazine. a. represents PAPA seawater. b. represents modified seawater. 5 ' J , Figure 14. The effect of pH on the photolytic reaction rate of xanthopterin. a. represents PAPA seawater. b. represents modified seawater. 99. Figure 15. The effect of pH on the photolytic reaction rate of isoxanthopterin. a. represents PAPA seawater. 100. Figure 16. Changes i n the absorption spectrum of dioxylumazine i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. Changes i n the absorption spectrum of isoxanthopterin i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. Changes i n the absorption spectrum of leucopterin i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. 103. Figure 19. Changes i n the absorption spectrum of oxylumazine i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. Changes i n the absorption spectrum of xanthopterin i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. 105. Figure 21. Changes i n the absorption spectrum of lumazine i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. Also, the absorption spectrum of lumazine at zero hours a f t e r a c i d i f i c a t i o n to pH 1.0. 106. Figure 2 2 . Changes i n the absorption spectrum of pt e r i n i n PAPA seawater with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. Also, the absorption spectrum of p t e r i n at zero hours a f t e r a c i d i f i c a t i o n to pH 1 . 0 . 107. Figure 23. Changes i n the absorption spectrum of oxylumazine i n pl-l 7.0 sodium phosphate buffer with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. .103. Figure 24. Concentration versus time for the photolytic decomposition of oxy-lumazine [OLlfJ and f o r the appearance of dioxylumazine [DIOLU] i n PAPA, synthetic and modified seawater under aseptic conditions. P A P A 100 200 TIME (hours) 109. Figure 25. Concentration versus time for the pho t o l y t i c decomposition of oxy-lumazine [OLlO and for the appearance of dioxylumazine [DIOLU] i n pH 10.0 (sodium carbonate) and pH 8.0 (sodium phosphate) buffers under aseptic conditions. TIME (hours) 110. Figure 26. Concentration versus time f o r the ph o t o l y t i c decomposition of oxy-lumazine [jOLU] and for the appearance of dioxylumazine [piOLlQ i n pH 7.0 (sodium phosphate) and pH 4.0 (sodium acetate) buffers under aseptic conditions. Buffer pH 7.0 Buffer pH4.0 TIME (hours) 111. Figure 27. Concentration versus time for the pho t o l y t i c decomposition of oxylumazine [OLU] and for the appearance of dioxy-lumazine QDIOLlf] i n pH 5.4 (sodium acetate) buffer under aseptic conditions. TIME (hours) 112. Figure 28. Changes i n the absorption spectrum of xanthopterin i n pH 7.0 sodium phosphate buffer with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps under aseptic conditions. 113. Figure 29. Absorption spectra of leucopterin and xanthopterin i n pH 8.0 phosphate buffer and a f t e r a c i d i f i c a t i o n to pH 1.0. Leucopterin pH 8.0 ( ) pH 1.0 ( ) xanthopterin pH 8.0 ( ) pH 1.0 ( ) 114. Figure 30. Concentration versus time f or the phot o l y t i c decomposition of xanthopterin QcPT~j and for the appearance of leucopterin QLPT] i n PAPA and synthetic seawater under aseptic conditions. Y — i — i — • — i — • — i — i — i > i — 1 — | — i — i — ' — i — i — i — " — r * ~ i — ' — | — ' — | — ' — | — i — | — " — | — i — p 0 200 400 600 800 1000 0 200 400 600 TIME (hours) 115. Figure 31. Concentration versus time f o r the ph o t o l y t i c decomposition of xanthopterin [ X P l f j and for the appearance of leucopterin [ L P T ] i n modified seawater and ion mix medium under aseptic conditions. 0.05 TIME (hours) 116. Figure 32. Concentration versus time for the photolytic decomposition of xanthopterin £XFT~] and for the appearance of leucopterin [jLPT] i n pH 10.0 (sodium carbonate) and pH 8.0 (sodium phosphate) buffer under aseptic conditions. TIME (hours) 117. Figure 33. Concentration versus time f o r the photolytic decomposition of xanthopterin Q\PT] and for the appearance of leucopterin [LPTT] i n pH 7.0 (sodium phosphate) and pH 4.0 (sodium acetate) buffers under aseptic conditions. TIME (hours) 113. Figure 34. Concentration versus time f o r the decomposition of xanthopterin [~XPT~] and for the appearance of leucopterin £ L P T ] i n pH 5.0 (sodium acetate) buffer under aseptic conditions. TIME (hours) igure 35. Absorption spectrum of isoxanthopterin i n pH 8.0 sodium phosphate buffer ( ) and a f t e r a c i d i f i c a t i o n to pH 1.0 ( •). 120. Figure 36. Temporal changes i n the absorption (E) of adenine, guanine, hypoxanthine, and xanthine i n N3M-medium under aseptic conditions while under i l l u m i n a t i o n (cool-white fluorescent lamps) or i n dark storage at room temperature. 1 ' 1 1 1 i r— ADENINE at 2 6 0 n m . i » r 0.55 0.5 0 H + H 1 V. GUANINE at 246 nm. 0.2 0 -0.1 5 -^ 1 1 1 (. 0.6 0 0.5 5 -0.50 - ' HYPOXANTHINE at 2 4 9 n m . dark H 1 H dark. H 1 h dark light H h XANTHINE at 272nm. 0.6 0 -0.5 5 0.5 0 --' i - i i • i • » 1 1 H dark light 100 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 TIME (hours) 700 121. Figure 37. Changes i n the absorption spectrum of adenine and guanine under aseptic conditions i n NSM-medium with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps or storage i n darkness at room temperature. The absorption spectrum of adenine and guanine (• •) a f t e r a c i d i f i c a t i o n to pH 1.0. 0.3-0.2-A d e n i n e d a r k A f ^  1 1 IJ ll 1 1 1 1 1 ll ! ' u j -0 Hours i 1 i i H-670 Hours i i i l A d e n i n e l ight -0 Hours -670 Hours Changes i n the absorption spectrum of. hypoxanthine and xanthine under aseptic conditions i n NSM-medium with the indicated periods of i l l u m i n a t i o n by cool-white fluorescent lamps or storage i n darkness at room temperature. The absorption spectrum of hypoxanthine and xanthine (» •) a f t e r a c i d i f i c a t i o n to pH 1 . 0 . 123. Figure 3 9 . Concentration versus time for the decomposition of u r i c a c id under aseptic conditions i n NSM-medium during i l l u m i n a t i o n by cool-white fluorescent lamps or storage i n darkness at room temperature. 124. Figure 40. Changes i n the absorption spectrum of u r i c acid under aseptic conditions i n NSM-medium with the indicated periods of i l l u m i n a t i o n (cool-white fluorescent lamps) or storage i n darkness at room temperature. The absorption spectrum of u r i c acid ( •) a f t e r a c i d i f i c a t i o n to pH 1.0. nm nm 125. Figure 41. Diagram of the xanthopterin molecular species and percentage hydrated expected at various pH values. (• ) pK determination curve. (•———•) or (? ?) percentage of hydrated species (of structure shown) present i n equilibrium with the non-hydrated species at the pH's shown. 126. Figure 42. Diagram of the oxylumazine molecular species and percentage hydrated expected at various pH values. ( ) piC determination curve. (? ?) estimated percentage of hydrated species (of structure shown) present i n equilibrium with the non-hydrated species at the pH's shown. 1.27. Figure 43. Proposed dark reaction scheme for the stoichiometric conversion of oxylumazine to dioxylumazine and xanthopterin to leticopterin. A - 1:1 stoichiometric oxidation of oxylumazine i n NaCl solutions. B - 2:1 stoichiometric oxidation of oxylumazine i n seawater. C - 1:1 stoichiometric oxidation of xanthopterin i n seawater and buffered media. (For r e l a t i v e rates of xanthopterin and leticopterin breakdown, see Table I X ) . 0 HN [NaCl] 0 -F ,0 ^ r O H , OXYLUMAZINE at p H 8 2 Cu 2+ COMPLEX 2 C u 2 4 I H OXYLUMAZINE<" On H£0 -HN ^ O ^ N ' H [0] ^ MKI /^V^ Seawater O ^ N X S H H H N B possible seawater anion promotion H 2 C u 2+ COMPLEX 2 C u 2 + 10XYLUMAZINE<^ H DIOXYLUMAZINE H / 2 C U ( + 2 H - ) XX X ' 2Cu 2 + 2 C u + v i V seawater ^/ degradation produces) unidentified NH^H anions ? (^ presumably pyrimidine derivatives) 0 N H ^ N^Q- f H a O •N1 -HgO ^ HN NH NvO~ 0 H XANTHOPTERIN (pH 7-10) IpN^N^OH f \ NH^vW)1 rXn' ^ n r0l HO L u j n^ y LEUCOPTERIN (pH 7-9) OR HN YY . ' T IM / \ > H i I H [0] HgO H XANTHOPTERIN (pH4.0) slow rxn. r— ZD Q O CC 0 . Q LU LU O LEUCOPTERIN (pH 4.0) 128. Figure 44. Diagram of the dioxylumazine molecular species expected at various pH values. ( ) pk" determination curve. Diagram of the leucopterin molecular species expected at various pH values ( ) pk" determination curve. 130. Figure 46. Diagram of the 6,7-dihydroxypteridine molecular species expected at various pH values. ( ) pk" determination curve. 131. Figure 47. Proposed and known fire-membered ring chelates between pteridine derivatives and divalent metal cations (M). Also, known complexes of benzenoid compounds involving vic i n a l hydroxyl groups and divalent metal cations. A - proposed (1:1) 6,7-dihydroxyl-pteridine complex. E - known (1:1) pteridine complex. C - known (2:1) pteridine complex. D - known (1:1) benzenoid complex. E - known (2:1) benzenoid complex. X" = - H catechol - C O O H protocatechuic acid -CH2COOH homoprotocatechuic acid - C H 2 C H 2 C O O H 3,4-dihydroxyhydrocinnamic acid 1 3 2 . Figure 48. Diagram of the isoxanthopterin molecular species expected at varioiis pH values. ( ) pk* determination c\j.rve. (- ) estimated pk* determination curve. 133. Figure 4 9 . Diagram of the molecular species of lumazine and p t e r i n expected at various pH values. ( ) pic" determination curve. Absorption spectra of 6-hydroxy-2 , 4 ,5-triaminopyrimidine ( ) cytosine ( ), pyrazinamide ( ) , and 2-pyrazinol ( ) i n PA-PA seawater. 135. LITERATURE CITED. Abbot, B . C . , and D. Bal lantyne . 1957. The tox in from Oymnodinlum veneficum B a l l a n t i n e . J . Mar. B i o l . Assoc. U . K . , 36: 169-189. A l b e r t , A. 1950. The a v i d i t y of f o l i c a c i d and other pter id ines of the ions of heavy metals . Biochem. J . , 47: IX-X. A l b e r t , A. 1953a. Quant i ta t ive studies of the a v i d i t y of n a t u r a l l y occurring substances for trace metals . 3. P t e r i d i n e s , r i b o f l a v i n , and pur ines . Biochem. J . , 54: 646-654. A l b e r t , A . 1953b. The p t e r i d i n e s . Quart . Rev. of the Chem. Soc . , 6; 197-237. A l b e r t , A. 1955. P t e r i d i n e s t u d i e s . Part V I I . The degrada-t i o n of 4 - , 6 - , and 7-hydroxypteridine .by ac id and a l k a l i . J . Chem. S o c , 2690-2699. A l b e r t , A. 1956. Photo-reduct ion of p t e r i d i n e s . Nature, 173: 1072. A l b e r t , A . 1959. Heterocyc l i c chemistry - An Introduct ion . U n i v e r s i t y of London, The Athlone Press , London, p. 38-132. A l b e r t , A . 1963. Ion izat ion Constants, In Phys ica l Methods i n Heterocyc l i c Chemistry, V o l . 1. , E d . A.P.. K a t r i t z k y , Academic Press , I n c . , New York. p. 2-108. A l b e r t , A . 1964. Covalent hydrat ion of p t e r i d i n e s . In P t e r i d i n e Chemistry, Ed . W. P f l e i d e r e r . Pergamon Press , New York. p . 111-128. A l b e r t , A . 1969. Personal communication. A l b e r t , A . 1970. Personal communication. A l b e r t , A . , and D . J . Brown. 1954. Purine s t u d i e s . Part I . S t a b i l i t y to ac id and a l k a l i , s o l u b i l i t y , i o n i z a t i o n . Comparison with p t e r i d i n e s . J . Chem. S o c , 2060-2071. A l b e r t , A . , and H . C . S . Wood. 1953. P ter id ine syntheses. I I . Isoxanthopterin. J , A p p l . Chem., 2: 521-523. A.lbert, A . , and H. Yamamoto. 1968a. The a c t i o n of ac id and a l k a l i on p t e r i d i n e . J . Chem. Soc. (C) , 2239-2292. A l b e r t , A . , and H. Yamamoto. 1968b. P t e r i d i n e s tudies . Part XXXV. The s tructure of the hydrated dimer formed by the act ion of d i l u t e a c i d on 4-methylDteridine . J . Chem. S o c (C) , 1181-1187. 136. A l b e r t , A., T.J. Batterham, and J . J. McCormack. 1966. Pteridine studies. Part XXXIII. E q u i l i b r a between 3,4-hydrated and 5,6,7,8-dihydrated cations. J . Chem. Soc.(B), 1105-1109. Albert, A., D.J. Brown, and G. Cheeseman. 1952a. Pteridine studies. I I . 6- and 7-hydroxylpteridines and t h e i r d e r i v a t i v e s . J. Chem. S o c , 1620-1630. Albert, A., D.J. Brown, and G. Cheeseman. 1952b. Pteridine studies. I I I . The s o l u b i l i t y and the s t a b i l i t y to hydrolysis of pt e r i d i n e s . J . Chem. Soc., 4219-4232. Albert, A., D.J. Brown, and H.C.S. Wood. 1956. Pteridine studies. Part VIII. The degradation of pt e r i d i n e . Methylation of the hydroxypteridines and degradation of the products. J . Chem. Soc., 2066-2075. Antia, N.J., and J.Y. Cheng. 1970. The s u r v i v a l of axenic cultures of marine planktonic algae from prolonged exposure to darkness at 20°C. Phycologia, 9: 179-183. Antia, N.J., and V. Chomey. 1968. Nature of the nitrogen compounds supporting phototrophic growth of the Marine cryptomonad Hemiselmis virescens. J . Protozoology, 15(1): 198-201. Armstrong, F.A.J., and G.T. Boalch. 1961. U l t r a - v i o l e t absorp-t i o n of seawater. Nature, 192: 858-859. Athavale, V.T., N. Mahadevan, and R.M. Sathe. 1968. Potentio-metric study of the complexes of catechol, t i r o n , and a - p i c o l i n i c a c i d with some metal ions. Indian J. Chem., 6: 660-662. Athavale, V.T., L.H. Prabhu, and D.G. Vartak. 1966. Solution s t a b i l i t y constants of some metal complexes of derivatives of catechol. J . Inorg. Nucl. Chem., 28: 1237-1249. Avis, P.G., F. Bergel, and R.C. Bray. 1956. C e l l u l a r c o n s t i -tuents. The chemistry of xanthine oxidase. Part III. Estimations of the co-factors and the c a t a l y t i c a c t i v i t i e s of enzyme fractions from cow's milk. J . Chem. S o c , 1219-1226. Baylor, E.R., and W.H. S u t c l i f f e , J r . 1965. Dissolved organic matter i n seawater as a source of p a r t i c u l a t e food. Limnol. Oceanog., 8; 369-372. Belser, W.L. 1963. Bioassay of trace substances. In The Sea. Vol. 1. Ed. M.N. H i l l . Interscience Publishers, New York, p. 220-231. 137. Bendich, A . 1955. Chemistry of Purines and Pyr imid ines . In The Nucle ic Ac ids , V o l . 1, Ed . E . Chargaff and J . N . Davidson. Academic Press , I n c . , New York. p. 81-136. Bent ley , J . A . i960. Plant growth hormones i n marine phyto-plankton, zooDlankton and seawater. J . Mar. B i o l . Assoc. U . K . , 39: 433-444. Bergmann, F . , and H. Kwietny. 1958. P ter id ines as substrates of mammalian xanthine oxidase. I . The end-product of the enzymic ox idat ion of p t e r i d i n e s . Biochim. Biophys. Acta , 28: 613-618. Bergmann, F . , and H. Kwietny. 1959. Pter id ines as substrates of mammalian xanthine oxidase. I I . Pathways and rates of ox ida t ion . Biochim. Biophys. A c t a , 33: 29-46. B irdsey , E . C . 1962. The ni trogen u t i l i z a t i o n and metabolism of u n i c e l l u l a r a lgae . A t h e s i s , Stanford U n i v e r s i t y . B l a k l e y , R . L . 1969. Natural occurrence of p ter ins and fo la te d e r i v a t i v e s . Chapter 2. In Volume 13 of F r o n t i e r s of B io logy , The Biochemistry of F o l i c A c i d and Related P t e r i d i n e s . North-Holland Publ ich ing C o . , Amsterdam, p . 8-57. Bray, R . C . 1963. Xanthine Oxidase. In The Enzymes, V o l . 7, Ch. 22, Eds. P . D . Boyer, H . Lardy, and K. MyrbSck. Academic Press , I n c . , New York. p . 533-556. Erown, D . J . , and S . F . Mason. 1956. P t e r i d i n e s t u d i e s . Part IX. The s tructure of the monohydroxypteridines and t h e i r N-methyl d e r i v a t i v e s . J . Chem. S o c , 3443-3453. Eurkholder , P.R. 1959. Vitamin-producing bac ter ia i n the sea. R e p r i n t , Internat ional Oceanographic Congress, New York, p. 912-913. C a n z a n e l l i , A . , R. G u i l d , and D. Rapport. 1951. Ammonia and urea product ion and changes i n absorpt ion spectra of n u c l e i c ac id der iva t ives fol lowing u l t r a v i o l e t i r r a d i a t i o n . Am. J . P h y s i o l . , 167: 364-374. C a v a l i e r i , L . F . , and A. Bendich. 1950. The u l t r a v i o l e t absorp-t i o n spectra of pyrimidines and pur ines . J . Am. Chem. S o c , 72: 2537-2594. Cheng, J . Y . , and N . J . A n t i a . 1970. Enhancement by g l y c e r o l of phototrophic growth of marine p lanktonic algae and i t s s i g n i f i c a n c e to the ecology of g l y c e r o l p o l l u t i o n . J . F i s h . Res. Bd. Canada, 27: 335-346. Chris tensen , E . , and A . C . Giese . 1954. Changes i n absorpt ion spectra of nuc l e i c ac ids and t h e i r der iva t ives fo l lowing exposure to ozone and u l t r a v i o l e t r a d i a t i o n . A r c h . Biochem. R i o p h . , 51: 208-216. 138. C l a r k , J . and G. Neath. 1966. Heterocyc l i c s tud ie s . Part I . Ring-opening of some 4-hydroxypteridine d e r i v a t i v e s . J . Chem. Soc. (C) , 1112-1116. C l a r k , J . , and G. Neath. 1968. Heterocyc l i c s tud ie s . Part IV. The ac t ion of hydroxylamine on 4-hydroxypteridine and i t s methyl d e r i v a t i v e s . J . Chem. Soc. (C) , 919-922, C l a r k , J . , and C. Smith. 1969. Heterocyc l i c s tud ie s . Part X I . Some 3-methoxypteridine-4(3H)-ones. J . Chem. Soc. (C) , 2777-2780. C l a r k , J . , G. Heath, and C. Smith. 1969. He terocyc l i c s tud ies . Part V I I . A c t i o n of methoxyamine and. methylhydrazines on 4-hydroxypteridine and i t s methyl d e r i v a t i v e s . J . Chem. Soc. (C), 1297-1301. C r a i g i e , J . 3 . , and J . KcLachlan . 1964. Excre t ion of co lored u l t r a v i o l e t - a b s o r b i n g substances by marine algae. Can. J . B o t . , 42: 23-33. Banforth , W.F. 1962. Substrate a s s i m i l a t i o n and heterotrophy. In "Physiology and Biochemistry of Algae". .Ed. R . A . Lewin. Academic Press , Inc . , New York, p . 99-123. Dawson, R . M . C . , D . C . E l l i o t , W.H. E l l i o t t , and K.M. Jones. 1969. Data for Biochemical Research, 2nd e d i t i o n , At the Clarendon Press , Cxford. 654 p . Dejens, E . T . , J . H . Reuter, and K . N . F . Shaw. 1964. Biochemical compounds i n offshore C a l i f o r n i a sediments and seawaters. Geochimica et Cosmochimica A c t a . , 28.: 45-66. Descimon, H. 1971. Les pte'rines des P ier idae (Lepidoptera) et l e u r biosynthese I - I d e n i t i f i c a t i o n des p r i n c i p a l e s pter ines des Col ias croceus (Fourcroy) et de quelques autres especes de P i e r i d a e . Biochimie , 53: 407-418. Droop, M.R. 1957. Vitamin B,? i n marine ecology. Nature, 180: 1041-1042. L Duursma, E . K . 1961. Disso lved organic carbon, n i t rogen , and phosphorous i n the sea. Netherlands J . Sea Research, 1.: 1-141. Duursma, E . K . 1965. The d i s so lved organic const i tuents of seawater. In Chemical Oceanography, V o l . 1, Ed . J . P . R i l e y and G. Skirrow. Academic Press , I n c . , New York, p. 433-475. Ewing, G.W. 1969. Instrumental methods of chemical a n a l y s i s . T h i r d E d i t i o n . McGraw-Hil l Book Company, New York. F e l l i g , J . 1954. Production of t r i u r e t from u r i c a c i d by u l t r a v i o l e t i r r a d i a t i o n . Science, 119: 129-130. 139. , F o r r e s t , H . 3 . , and H.K. M i t c h e l l . 1954. Pter id ines from D r o s n h i l i a . I I . Structure of the yellow pigment. J . Am. Chem. S o c . , 16: 5658-5662. F o r r e s t , H . S . , C. Van Baalen, and J . Myers. 1957. Occurrence of p ter id ines i n a blue-green a l g a . Science, 125: 699-700. Fox, D . L . i960. Perspectives i n marine biochemistry . Annals New York Acad. Science, 90: 617-621. F u j i i , R. 1969. Chromatophores and pigments. In F i s h Physiology, V o l . 3, Ed . W.3. Hoar and D . J . R a n d a l l . Academic Press , I n c . , New York. p. 307-353. F u l l e r , R . C . , and N.A. Nugent. 1969. P ter id ines and the funct ion of the photosynthetic r eac t ion center . Proc . Nat. Acad. Sc. U . S . A . , 63(4): 1311-1318. Garay, K . , and F . Guba. 1954. Die Revers ib le Veranderung des Adenosintriphosphates in fo lge von Strahlungswirkungen. Acta P h y s i o l . Acad. Sc . Hung., 5: 393. Gomori, G . 1955. Preparat ion of buffers for use i n enzyme s tud ies . In Methods i n Enzymology, V o l . 1, Ed . S . P . C . Colowick and N.O. Keplan. Academic Press , I n c . , New York, p . 138-146. G r i f f i t h s , M. 1952. Oxidat ion of u r i c ac id cata lyzed by copper and by the cytochrome-cytochrome oxidase system. J . B i o l . Chem., 197: 399-407. Harvey, H.W. 1955. The Chemistry and F e r t i l i t y of Sea Waters. Cambridge U n i v e r s i t y Press , London, p.151-152. H a t f i e l d , D . L . , C. Van Baalen, and H . 3 . F o r r e s t . 1961. P t e r i -dines i n blue-green a lgae . Plant P h y s i o l . , .36: 240-243. He l l ebus t , J . A . 1965. Excre t ion of some organic compounds by marine phytoplankton. Limnol . Oceanog., 10: 192-206. Holm-Hansen, 0 . , J . D . H . S t r i c k l a n d , and P.M. Wi l l iams . 1966. A d e t a i l e d analys i s of b i o l o g i c a l l y important substances i n a p r o f i l e of f southern C a l i f o r n i a . Limnol . Oceanog., JUL: 548-561. Hood, D.W. 1963. Chemical Oceanography. Oceanography and marine b i o l o g y . Annual Review, V o l . 1, E d . H. Barnes, p . 129-155. Home, R.A. 1969. Marine Chemistry Wi ley - Intersc ience , New York. p . 199 and 274. 140. Hunt, J . M . 1962. Some observations on organic matter i n sediments. Paper presented at the S c i e n t i f i c Sess ion, 25 years of Hungarian O i l , Budapest. Inoue, Y . , and D.D. P e r r i n . 1962. P t e r i d i n e s t u d i e s . Part XX. Revers ib le water a d d i t i o n to hydroxypter id ines . J . Chem. S o c , 2600-2606. Inoue, Y . , and D.D. P e r r i n . 1963a. P t e r i d i n e s tud ie s . Part X X I I I . K ine t i c s of the r e v e r s i b l e hydrat ion of 6-hydroxy-p t e r i d i n e and some d e r i v a t i v e s . J . Chem. S o c , 4803-4806. Inoue, Y . , and D.D. P e r r i n . 1963b. P t e r i d i n e s tud ie s . Part XXI. K ine t i c s of the r e v e r s i b l e a d d i t i o n of water t o , and r ing-opening of, p t e r i d i n e and i t s methyl d e r i v a t i v e s . J . Chem. S o c , 2648-2654. Jacobsen, N.W. 1966. P t e r i d i n e s tud ie s . Part XXXI. The covalent hydrat ion and subsequent ox idat ion of 8-methyl der iva t ive s of some amino- and hydroxy-pter id ines . J . Chem. S o c ( C ) , 1065-1072. J e f f r e y , L . M . , and D.W. Hood. 1958. Organic matter i n seawater; an eva luat ion of various methods for i s o l a t i o n . J . Mar. R e s . , 17: 247-271. Kauffmann, T . 1959. Notiz uber die K u n s t i t u t i o n des Ichthyo-p t e r i n s . L ieb igs Annalen der Chemie, 625: 133-139. Kland, M . J . , and L . A . Johnson. 1957. A k i n e t i c study of the u l t r a v i o l e t decomposition of biochemical der iva t ives of n u c l e i c a c i d . I . Pur ines . J . Am. Chem. S o c , 79: 6187-6192. K o l l e r , L . R . 1965. U l t r a v i o l e t Radiat ion,2nd E d i t i o n , John Wiley and Sons Inc . , New York. p . 130-134. K u s h i b i k i , K. , T . Hama, and T . Goto. 1954. Ouelques pter ines i so lees de l a peau ou des e c a i l l e s de l a Carpe et l e u r transformation photochimique. Comptes Rendus, Societe de B i o l o g i e , 148: 759-762. Kwietny, H . , and ?. Bergmann. 1959. Separation and i d e n t i f i c a -t i o n of p ter id ines by paper chromatography. J . Chrom., 2: 162-172. Lee, A . S . K . , W.E. Vanstone, J . R . Markert , and N . J . A n t i a . 1969. UV-absorbing and UV-f luoresc ing substances i n the b e l l y s k i n of f ry of Coho Salmon (Oncorhynchus k i s u t c h ) . J . F i s h . Res. Bd. Canada, 26(5): 1185-1198. Levenberg, B . , and 0 . H a y a i s h i . 1959. A b a c t e r i a l p t e r i n deaminase. J . B i o l . Chem., 234(4): 955-961. Levy, C . C . , and W.S. McNutt. 1962. The b i o l o g i c a l transforma-t i o n of xanthopterin by a bacterium i s o l a t e d from s o i l . B iochemis try ,1 (6 ) : 1161-1170. 141. L i t c h f i e l d , C.D., and D.W. Hood. 1966. M i c r o b i o l o g i c a l assay f o r orgnic compounds i n seawater. Apo. M i c r o b i o l . , 14(2): 145-151. Loofbourow, J.R. 1948. E f f e c t s of u l t r a v i o l e t r a d i a t i o n on c e l l s . Growth Symposium, Supplement t o V o l . X I I . p. 75-149. Loofbourow, J.R., and M.M. Stimson. 1940. U l t r a v i o l e t s p e c t r a of nitrogenous h e t e r o c y c l i c compounds. F a r t I. E f f e c t of p!i and i r r a d i a t i o n on the spectrum of adenine. J . Chem. Soc., 844-848. Lyman, J . , and R.H. Fleming. 1940. Composition of seawater. J . Mar. Res., 3: 134-146. M c A l l i s t e r , C.D., T.R. Parsons, and J.D.H. S t r i c k l a n d . i960. Primary p r o d u c t i v i t y at s t a t i o n "P" i n the north-east P a c i f i c Ocean. E x t r a i t du J o u r n a l du C o n s e i l I n t e r n a t i o n a l Pour L ' E x p l o r a t i o n de l a Mere, 25(3): 240-259. McCormack, J . J . , and D.M. V a l e r i n o . 1970. E f f e c t s of p t e r i d i n e s and r e l a t e d compounds on xanthine oxidase i n . v i t r o . In Chemistry and B i o l o g y of P t e r i d i n e s , Eds. X. Iwai, M. Goto, M. Akino and Y. Iwanami, I n t e r n a t i o n a l Academic P r i n t i n g Co., L t d . , Tokyo. p. 381-389. McNutt, W.S. 1962. The enzymatic conversion of p t e r i d i n e s i n t o x a n t h i n e - 8 - c a r b o x y l i c a c i d . In P t e r i d i n e Chemistry. Ed. W. P f l e i d e r e r , Pergamon Press, New York. p. 427-441. M o r i , Y., J . Matsumoto, and T. Hama. 1960. On the p r o p e r t i e s of cyprino-pourpre A2, a p t e r i n i s o l a t e d from the s k i n of C y p r i n i d a e , and i t s r e l a t i o n t o i c h t h y o p t e r i n or 7-hy d r o x y b i o p t e r i n . Z e i t s c h r i f t f u r vergleichende P h y s i o l o g i e , 43: 531-543. Nawa, S. 1960. The s t r u c t u r e of the y e l l o w pigment from D r o s o p h i l a . B u l l . Chem. Soc. Japan, 33(11): 1555-1560. O'Dell, B.L.,J.M. Vandenbelt, E.S. Bloom, and J . J . P f i f f n e r . 1947. Hydrogenation of v i t a m i n B ( p t e r o y l g l u t a m i c a c i d ) and r e l a t e d p t e r i d i n e s . J . Am. Chem. Soc., 69: 250-253. Ogura, M. 1970. On the presence of 0.1-0.5-^1 d i s s o l v e d organic matter i n seawater. Limnol. Oceanog., JL5_: 476-479. Parsons, T.R. 1963. Suspended organic matter. In Progress i n Oceanography, V o l . 1, Ed. M. Sears. MacMillan, New York, p. 203-239. Parsons, T.R., and J.D.H. S t r i c k l a n d . 1962. On the prod u c t i o n of p a r t i c u l a t e organic carbon by h e t e r o t r o p h i c processes i n seawater. Deep-Sea Research,. 8: 211-222. 142. P e r r i n , D.D. 1962. P t e r i d i n e s t u d i e s . Part XVI. E q u i l i b r i a i n aqueous s o l u t i o n s of p t e r i d i n e . J . Chem. 3oc., 645-653. P f l e i d e r e r , W. 1963. The s o l u b i l i t y of h e t e r o c y c l i c compounds. In P h y s i c a l Methods i n H e t e r o c y c l i c Chemistry, V o l . 1, Ed. A.R. X a t r i t z k y . Academic Pre s s , Inc., New York, p. 177-188. P l u n k e t t , M.A., and N.W. Rakestraw. 1955. D i s s o l v e d organic matter i n the sea. Deep-Sea Research, Supplement, 3: 12-14. P o c k l i n g t o n , R. 1971. Free amino-acids d i s s o l v e d i n North A t l a n t i c Ocean Waters. Nature, 230: 374-375. Poole, H.H., and W.R.G. A t k i n s , 1937. The p e n e t r a t i o n i n t o the sea of l i g h t of v a r i o u s wave-lengths as measured by emission or r e c t i f i e r p h o t o - e l e c t r i c c e l l s . Proc. Royal Soc. London, Series B, 123: 151-165. P r o v a s o l i , L. 1963. Organic r e g u l a t i o n of phytoplankton f e r t i l i t y . In the Sea, V o l . 1, Ed. M.N. H i l l . I n t e r -science P u b l i s h e r s , New York. p. 165-219. Rembold, H. 1970. Catabolism of unconjugated p t e r i d i n e s . In Chemistry and B i o l o g y of P t e r i d i n e s . Eds. K. Iwai, M. Akino, M. Goto and Y. Iwanami. I n t e r n a t i o n a l Academic P r i n t i n g Co., L t d . , Tokyo, p. 163-178. Rembold, H., and F. Simmersbach. 1969. Catabolism of p t e r i d i n e c o f a c t o r s . I I . A s p e c i f i c p t e r i n deaminase i n r a t l i v e r . Biochim. Biophys. A c t a , 134: 589-596. Rembold, H. , H. Metzger, and W. Gutensohn. 1971. Catabolism of p t e r i d i n e c o f a c t o r s . I I I . On the i n t r o d u c t i o n of an oxygen f u n c t i o n i n t o p o s i t i o n 6 of the p t e r i d i n e r i n g . Biochim. Biophys. A c t a , 230: 117-126. Rembold, H. , H. Metzger, P. Sudershan, and W . Gutensohn. 1969. Catabolism of p t e r i d i n e c o f a c t o r s . I. P r o p e r t i e s and metabolism i n r a t l i v e r homogenates of t e t r a h y d r o b i o p t e r i n and t e t r a h y d r o n e o p t e r i n . Biochim. Biophys. Acta, 184: 336-396. R i l e y , G.A. 1953. A L e t t e r t o the E d i t o r . J o u r n a l Du C o n s e i l , 19: 85-39. Rohrscheid, F., A.L. B a l c h , and R.H. Holm. 1966. P o t e n t i a l e l e c t r o n t r a n s f e r complexes of the type: synthesis and p r o p e r t i e s of complexes derived from pyrocatechol and t e t r a c h l o r o p y r o c a t e c h o l . Inorg. Chem., 5: 1542-1551. Rowan, T., and H.C.S. Wood. 1968. The b i o s y n t h e s i s of p t e r i d i n e s . Part V. The sy n t h e s i s of r i b o f l a v i n from p t e r i d i n e p r e c u r s o r s . J . Chem. Soc. (C), 452-458. 143. Rowan, T., H.C.S. Wood, and P. Hemmerich. 1961. N u c l e o p h i l i c a d d i t i o n s of 3 - s u b s t i t u t e d p t e r i d i n e s and t h e i r b i o -chemical s i g n i f i c a n c e . Proc. of the Chem. Soc., p. 260-261. Schou, M.A. 1950. Tautomeric conversion of x a n t h o p t e r i n . Arch. Biochem., 28: 10-29. Shaw, E., CM. Baugh, and C.L. Krumdieck. 1966. The chemical degradation of f o l i c a c i d . P h o t o l y s i s of 2 , 4 , 7 - t r i -h y d r o x y p t e r i d i n e . J . B i o l . Chem., 241(2): 379-382. Sober, H.A. 1968. Handbook of bioche m i s t r y , The Chemical Rubber Co., Cleveland, Ohio. S t a h l , P.H. 1969. The product of the a i r o x i d a t i o n of u r i c a c i d . An intermediate formed i n the presence of dim e t h y l -amine. Biochemistry, .8(2): 733-736. Steemann, N.E. 1955. Carbon d i o x i d e as a carbon source and n a r c o t i c i n photosynthesis and growth of Chiore11a pyrenoidosa. P h y s i o l . Plantarium,.8: 317-335. Stokstad, E.L.R., B.L. Kutchings, J.H. Mowat, J.H. Boothe, C.W. Waller, R.B. Angier, J . Semb, and Y.J. Subbarow. 1948. The degradation of the fermentation L a c t o b a c i l l u s  c a s e i factor. I. J . Am. Chem. Soc. , 70: 5-9. S t r i c k l a n d , J.D.H. 1958. S o l a r r a d i a t i o n p e n e t r a t i n g the ocean. A review of requirements, data, and methods of measurement, w i t h p a r t i c u l a r r eference t o p h o t o s y n t h e t i c p r o d u c t i v i t y . J . F i s h . Res. Bd. Canada, 15(3): 453-493. S t r i c k l a n d , J.D.H., and T.R. Parsons. 1968. A P r a t i c a l Handbook of Seawater A n a l y s i s . B u l l e t i n 167. F i s h e r i e s Research Board of Canada, Queen's P r i n t e r and C o n t r o l l e r of S t a t i o n e r y , Ottawa, Canada. S t u a r t , A., D.W. West, and H.C.S. Wood. 1964. P t e r i d i n e d e r i v a t i v e s . P a r t IX. 2,6~diamino~4-bydroxypteridine and r e l a t e d d i h y d r o p t e r i d i n e s . J . Chem. S o c , 4769-4774. Syl v a n i a B u l l e t i n . S y l v a n i a E l e c t r i c (Canada.) L t d . Engineering F u l l e t i n 0-283. S p e c t r a l energy d i s t r i b u t i o n curves of S y l v a n i a F40T12 f l u o r e s c e n t lamps. Commercial Engineering Department, Montreal, Quebec. 6 pages. S y r e t t , P.J. 1962. Nitrogen a s s i m i l a t i o n . In Physiology and Biochemistry of Algae. Ed. R.A. Lewin. Academic P r e s s , Inc., Mew York. p. 171-188. Tatewaki, M., and L. P r o v a s o l i . 1964. Vitamin requirements of three species of Antithamnion. Botanica Marina, 6: 193-203. 144. Tyson, C . A . , and A . E . K a r t e l l . 1968. E q u i l i b r i a of metal ions with pyrocatechol and 3 , 5 - d i - t - b u t y l - p y r o c a t e c h o l . J . Am. Chem. Soc . , 9 0 : 3379-3386. Va l l entyne , J . R . 1957. The molecular nature of organic matter i n lakes and oceans, with l e s s e r reference to sewage and t e r r e s t r i a l s o i l s . J . F i s h . Res. Bed. Canada, 14: 33-82. V i s c o n t i n i , M . , M. Schoe l l er , E . Loeser , P. Loeser, P. Karrer , and E . Hadorn. 1955. I so l ierung f luoresz i erender Stoffe aus Drosophi la melano^aster. He lvet ica Chimica A c t a , 38: 397-402. White, A . , P. Handler, and E . L . Smith. 1968. In P r i n c i p l e s of Eiochemistry , 4th E d i t i o n , McGraw-Hil l Book C o . , New York, p . 627-632. II (> Wieland, H . , and R. Purrmann. 1939. Uber die Flugelpigmente der Schmetterl inge . . IV. Die Beziehungeh zwischen xanthopterin und Leukopter in . L ieb igs Annalen der Chemie, 539: 179-187. Wieland, H . , and R. Purrmann. 1940. Uber die Flugelpigmente der Schmetterl inge. VI Uber Leukopterin und Xanthopter in . Liebigs Annalen der Chemie, 544: 163-182. Wieland, H . , and C. Schopf. 1925. Uber den gelben F l u g e l f a r b s t o f f des C i t ronenfa l t er s (Gonepteryx rhamni). Chemische Ber i ch te , 58: 2178-2183. Wieland, H. , H. Metzger, C. Schopf, and M. Pulow. 1933. Tiber Leukopter in , das Flugelpigment der Kohlweiji l inge ( P i e r i d e n ) . I I . L ieb igs Annalen der Chemie, 507: 226-265. Wood, S . J . F . 1958. The s i g n i f i c a n c e of marine microbio logy . B a c t e r i o l o g i c a l Reviews, 22: 1-19. Wood, E . J . F . 1965. Marine M i c r o b i a l Ecology. Reinhold Publ i sh ing Corporat ion , Mew York. 243 p . Z i e g l e r , I . 1962. Uber Natur l i ch Vorkommende Tetrahydro-p t e r i d i n e . In P t e r i d i n e Chemistry. E d . W. P f l e i d e r e r , Pergamon Press , New York. p . 295-305. APPENDIX A. DEFINITIONS autotroph - an organism capable of u t i l i z i n g inorganic materials and incapable of u t i l i z i n g organic materials, •facultative - optional, or, more accurately, capable of exercising an option t o . •heterotroph - an organism capable only of u t i l i z i n g organic compounds as a source of carbon, and energy, obligate - having only one l i f e condition, as distinguished from f a c u l t a t i v e , •phototrophic - any mode of n u t r i t i o n requiring l i g h t as a source of energy or fo r a key l i f e process, •pteridines - a class of organic compounds containing a carbon skeleton made up of fused pyrimidine and pyrazine rings as shown below. I 8 The t r i v i a l names and corresponding chemical names of the pteridines used i n t h i s study are given below. dioxylumazine - 2,4,6,7-tetrahydroxypteridine isoxanthopterin - 2-amino-4, 7-dihydroxypteridine leucopterin - 2-amino-4,6,7-trihydroxypteridine lumazine - 2,4-dihydroxypteridine oxylumazine - 2,4,6-trihydroxypteridine p t e r i n - 2-amino-4-hydroxypteridine xanthopterin - 2-amino-4,6-dihydroxypteridine APPENDIX A (contd.) -purines - a class of organic compounds containing a carbon skeleton made up of fused pyrimidine and imidazole rings as shown below. The t r i v i a l names and corresponding chemical name of the purines used i n thi s study are given below, adenine - 6-aminopurine guanine - 2-amino-6-hydroxypurine hypoxanthine - 6-hydroxypurine u r i c a c i d - 2,6,8-trihydroxypurine xanthine - 2,6-dihydroxypurine 147. APPENDIX B. EXPERIMENTAL MEDIA Taken from Lyman and Fleming (1940) and modified to experimental requirements. Synthetic Seawater^ 2) Modified Seawater Ion Mix Component : NaCl 19.55 gm 19.55 gm -MgCl 2-6H 2o( 3) 8.88 gm - 8.88 gm NaoS0, 2 4 3.265 gm 3.265 gm -CaCl 2-2H 20 1.220 gm . - 1.220 gm KC1 0.554 gm 0.554 gm mm NaHC03 0.1604 gm 0.1604 gm 0.l604gm KBr 0.080 gm 0.080 gm -H3BO3 0.0217 gm 0.0217 gm -SrCl 2•6H 20 0.0333 gm - -NaF 0.0025 gm 0.0025 gm -pH 8.0 ± ' 0.1 8.0 i 0.2 8.0 ± 0.1 Glass d i s t i l l e d water to 1000 ml. No trace metals were added to the above media. (1) a l l components were 'Baker Analyzed' reagent grade from J.T. Baker Chemical Co., P h i l l i p s b u r g , N.J. (2) s a l i n i t y of 21.5%a. (3) dried overnight i n a non-evacuated dessicator with phosphorous pentoxide. 148, APPENDIX B (contd.) NSM-Medium (used for purine studies only) Taken from Antia and Chorney (1968) and modified for experimental requirements. N aH 2P0 4«H 20 6.9 mg (50 umoles Na 23i0 3«9H 20 84.0 mg (300 pmoles Vitamins: Thiamine-HC1 0.5 mg (1.48 jumoles B i o t i n 0.001 mg (4.1 jimoles B12 0.002 mg (1.34 umoles Trace Metals (chelated): Na2EDTA*2H20 8.1 mg (21.8 umoles FeCl 3.6H20 2.7 mg (10.0 umoles MnS0 4«4H 20 1.125 mg (5.0 umoles ZnS0 4-7H 20 0.575 mg (2.0 umoles Na 2Mo0 4'2H 20 0.243 mg (1.0 umoles CuS0 4'5H 20 0.025 mg (0.1 umoles CoS0 4«7H 20 0.014 mg (50.0 umoles Buffer: TRIS.HC1 (4l.3mM,pH 6.8-6.9 before autoclaving) PAPA seawater, s a l i n i t y 33%0, to 1 l i t e r . 200 ml (lgm or 8.3 mmoles TRIS) This gives a pH of 7.6-7.8 i n the medium a f t e r autoclaving. Trace metals and PO^-SiO^ additions were 'Baker Analyzed' reagent grade chemicals from J.T. Baker Chemical Co., P h i l l i p s b u r g , N.J APPENDIX B (contd.) "PAPA" Seawater This seawater, nicknamed "PAPA", was obtained from the Ocean Weather Station "P" or "PAPA" at p o s i t i o n 50°N, 145°W i n the north-eastern P a c i f i c Ocean ( M c A l l i s t e r et a l . , 1960). The s a l i n i t y was given as 3 3 % 0 ( M c A l l i s t e r et a l . , i960) and the pH determined t o be 8.0 — 0.1. APPENDIX C. 150. ROUTINE STERILITY CHECKS The t e c h n i q u e of enrichment of p o s s i b l e m i c r o b i a l contami-nants ( b a c t e r i a and molds) by h e t e r o t r o p h i c growth i n a seawater medium e n r i c h e d w i t h o r g a n i c m a t e r i a l was used f o r checking r e a c t i o n mixtures f o r such c o n t a m i n a t i o n . The STP and ST 3 media of Tatewaki et a l . , (1964) were used f o r such c o n t a m i n a t i o n checks. One t o two drops of r e a c t i o n mixture were added t o a tube of each medium and i n c u b a t e d about t h r e e weeks i n t h e dark at room temperature (22-25°C) f o r a c o n t a m i n a t i o n check. The appearance i n e i t h e r medium of c l o u d i n e s s and t u r b i d i t y t y p i c a l of b a c t e r i a l growth or of d i s c o l o u r a t i o n and t u f t - f o r m a t i o n t y p i c a l of mold growth was t a k e n as evidence of p o s s i b l e c o n t a m i n a t i o n . From a p o s i t i v e tube, 1-2 drops were s u b c u l t u r e d i n the same media, f o r c o n f i r m a t i o n , and 1-2 drops were examined m i c r o s c o p i c a l l y . In the absence of any v i s i b l e changes i n t h e s t e r i l i t y check tube, the r e a c t i o n m i x t u r e t e s t e d was c o n s i d e r e d t o be f r e e from c o n t a m i n a t i o n . STERILITY TEST MEDIA Component: Seawater ( H A - M i l l i p o r e R f i l t e r e d ) D i s t i l l e d Water S o i l E x t r a c t KaN03 Ic^HPO ®a2 g l y c e r ° p h ° s p h a t e ^ ^ STP ST 3 800 ml 700 ml 150 ml 250 ml 50 ml 50 ml 200 mg 50 mg 10 mg 10 mg 151, APPENDIX C (contd.) Component: STP ST3 (2) 1-Iy-Case Amino - 20 mg Yeast Hydrolysate^ 1^ 200 mg Yeast E x t r a c t ^ - 10 mg (4) Dehydrated L i v e r Infusion - 20 mg Vitamin M i x ^ 1 ml 1 ml Vitamin 0.1 ;ug Sucrose 1 gm Carbon M i x ^ - 20 ml NaH-glutamate 200 mg DL-alanine 100 mg T r y p t i c a s e ^ 200 mg Glycine 100 mg G l y c y l g l y c i n e ^ - 400 mg pH 7.5-7.6 7.9 (1) N u t r i t i o n a l Biochemical Corporation, Cleveland, Ohio. (2) Acid digest of casein. S h e f f i e l d Chemical, a D i v i s i o n of National Dairy Products Corp., Norwich, N.Y. ( 3 ) Difco Laboratories Incorporated, D e t r o i t , Michigan. (4) Aqueous extract of Ox L i v e r , Oxo Ltd., London, England. (5) Pancreatic digest of casein, Baltimore B i o l o g i c a l Laboratory, Baltimore, Maryland. (6) Mann Research Laboratories Inc., New York, N.Y. 152. APPENDIX C (contd.) ( A) 1 ml Vitamin Mix Contains Thiamine-HCl 0.2 mg Pa.boflavin 5.0 ug N i c o t i n i c acid 0.1 mg Pyridoxine'2HCl 0.04 mg Putrescine'2HCl 0.04 mg Pyridoxamine•2HC1 0.02 mg Ca pantothenate 0.1 mg Choline H 2 C i t r a t e 0.01 mg B i o t i n 0.5 u § p-aminobenzoic a c i d 0.01 mg I n o s i t o l 1.0 mg F o l i c a c i d 2.5 PS Thymine 0.8 mg F o l i n i c a c i d 0.2 PS Orotic acid 0.26 mg Vitamin B.^ 0.05 (B) Carbon Mix Glycine 100 mg DL-alanine 100 mg L-asparagine 100 mg d i s t i l l e d water t o 100 Na acetate'3H 20 200 mg Glucose 200 mg L-glutamic a c i d 200 mg The s t e r i l i t y t e s t media were membrane-filtered ( M i l l i p o r e HA, 0.45-micron pore size) and transferred as 2.5 ml aliquots into 13.5 ml screw capped culture tubes. These tubes were then autoclaved for 15 min. at 120°C and 15 p s i , cooled, and stored i n a r e f r i g e r a t o r at 0-4°C. The tubes were allowed to warm to room temperature before use. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0101496/manifest

Comment

Related Items