Amino acids in suspended particulate matter from oceanic and coastal waters of the Pacific

215

Marine Chemistry, 6(1978) 215--231 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

AMINO ACIDS IN SUSPENDED PARTICULATE MATTER FROM OCEANIC AND COASTAL WATERS OF THE PACIFIC R. J. S I E Z E N * and T. H. M A G U E * *

Institute of Marine Resources, University o f California at San Diego, La Jolla, Calif. 92093 (U.S.A.) (Accepted December 1, 1977)

ABSTRACT Siezen, R. J. and Mague, T. H., 1978. Amino acids in suspended particulate matter from oceanic and coastal waters of the Pacific. Mar. Chem,, 6: 215--231. The concentration of 15 amino acids in hydrolyzed particulate matter from different regions and depths of the Pacific Ocean has been measured by gas--liquid chromatography. The relative composition was similar for all samples in the euphotic zone, where the particulate amino acid (PAA) concentration ranged from 370 to 2260 nmoles/l in coastal waters and from 90 to 260 nmoles/1 in the open ocean. Total PAA concentration dropped rapidly with depth, levelling off at 10--40 nmoles/1 below 200 m. Glycine, serine, glutamic acid and aspartic acid were the most abundant PAA in deep equatorial water and in deep off-shore California water. The nitrogen content of PAA could often account for 100% of the total particulate organic nitrogen present, while PAA carbon contributed up to 50% of the total particulate organic carbon in euphotic waters and down to 20% in deep waters. The protein equivalent to the total PAA content of particulate matter in near-surface waters amounted to 11--32 pg/1 at oceanic stations and up to 270 pg/1 at coastal stations.

INTRODUCTION

Suspended particulate organic matter (POM) in the oceans is an important c o m p o n e n t of the marine food web. In order to understand the pathways of material and energy flux during transformation and utilization of POM, a detailed knowledge of its organic constituents is required. There now exist abundant data on the spatial distribution of particulate organic carbon (POC) and particulate organic nitrogen (PON) in the oceans (Holm-Hansen et al., 1966; Menzel and Ryther, 1970; Chester and Stoner, 1974), b u t information is lacking on the specific molecular composition of POM. The protein content of POM has usually been estimated as PON x 6.25 * Present address: John Curtin School of Medical Research, Australian National University, Department of Physical Biochemistry, Canberra, A.C.T., Australia.

** Present address: Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575, U.S.A.

216

(Parsons and Strickland, 1962; Holm-Hansen, 1972) under the assumption that the a m o u n t o f non-protein nitrogen in POM is relatively insignificant. Packard and Dortch (1975) have measured actual protein in POM of North Atlantic waters, using a fluorescamine reaction specific for primary amino groups, and have in turn calculated the protein-nitrogen content. Although the assumption of the interconvertability of PON and protein has been supported by a direct comparison of the total nitrogen and amino-acid (unseparated, ninhydrin-positive substances) content of POM (Handa, 1970), these calculations provide little information a b o u t the constituents of this protein. Perhaps significant changes occur in the relative amino-acid composition of proteinaceous material during sinking from the euphotic zone or in areas of differing productivity. Degens et al. (1964) and Degens (1970) have provided preliminary information on the particulate amino-acid (PAA) content of POM, using two-dimensional paper chromatography and ion exchange c o l u m n c h r o m a t o g r a p h y followed by reaction with ninhydrin. They have shown that in the Sargasso Sea the relative percent composition of serine, alanine and cystine residues decreases with depth, while that of glycine, arginine and lysine increases. In order to assess in greater detail the possibility of significant compositional changes in the amino-acid content of POM, we have measured the concentrations of 15 c o m m o n amino acids in POM from different regions and depths of the Pacific Ocean. The analyses have been made by gas chromatography of the N-heptafluorobutyryl, ~O-isobutyl derivatives of amino-acid residues in hydrolysates of the particulate material, a method with the advantages of high sensitivity and the ability to distinguish certain u n c o m m o n amino acids which may serve as markers of specific algal groups. We have compared these results to the total PON and POC in replicate samples, as measured by a micro-Dumas high-temperature combustion. The total protein content of POM has also been estimated from the sum of the individual amino acid-residues.

METHODS AND M A T E R I A L S

Sample collection Water samples were collected in 30-1 PVC Niskin bottles fitted with tefloncoated closing springs. Samplers were cleaned before use with 70% 2-propanol. Immediately after collection, water samples were drained into glass carboys through a short length of silicone-rubber tubing covered on one end with 183 ~m-mesh Nitex netting (for removal of occasional large debris). Particulate material from 2 to 6 liters of water was collected on 24 or 42.5 mm Whatman GF/C glass fiber filters which had been baked in a muffle furnace at 450°C for 2 h. The filter-holders were borosilicate glass (Millipore) and differential pressure across the filter did not exceed 0.3 atm. Filters were transferred to small test tubes and stored frozen at --20°C until analysis.

217

Amino acid analysis Filters containing POM and added internal standard (norleucine) were hydrolyzed under vacuum in doubly-distilled 6N HC1 for 20 h at 110°C. The hydrolysates were filtered to remove disintegrated filter fibers, then dried on a rotary evaporator at 50°C and re-dissolved in 0.1N HC1. This material was applied to the top of a 5 mm x 50 mm column of Biorad AG 50W-X8 cation exchange resin previously cleaned with 2N NaOH, regenerated to the H + form with 2N HC1, and rinsed to neutrality with doubly-distilled water. After washing with three 0.5-ml portions of water, the amino acids were eluted with 4 ml of 5N NH4OH, collected in a small reaction vial, and evaporated to dryness under vacuum. The conditions for desalting by an ion-exchange column must be precisely controlled to prevent loss of acidic amino acids during washing and to assure recovery of the basic amino acids during elution. The N-heptafluorobutyryl, isobutyl esters of the amino acid samples were prepared by the m e t h o d of MacKenzie and Tenaschuk (1974) as modified by Siezen and Mague (1977); esterification was accomplished with 2-methyl-1propanol--3N HC1 and acylation with acetonitrile--heptafluorobutyric anhydride. The resulting derivatives were dissolved in ethyl acetate for analysis by flame-ionization gas chromatography. A Varian Aerograph 1400-10 instrument equipped with a 6 m x 2 mm i.d. glass column packed with 3% OV-101 on Gas Chrom Q, 8 0 - 1 0 0 mesh, was used. A temperature-programmed analysis from 80 to 250°C required 30 min to elute all the amino-acid derivatives. Quantification of peak areas was performed with a Hewlett-Packard 3373 B integrator. Reproducibility with standard mixtures of amino acids taken through the complete procedure of hydrolysis and derivatization was within 1--8% except aspartic acid, which was 11%. Arginine, histidine and cysteine/cystine were not reproducible, due to incomplete derivatization or decomposition on the chromatographic column. The total particulate carbon and nitrogen in replicate samples collected concomitantly with PAA samples were analyzed on a Hewlett-Packard model 185-B carbon--hydrogen--nitrogen analyzer as described by Sharp (1974). All glassware was either cleaned with chromic acid solution or baked at 450°C for 2--4 h before use to remove trace organic contamination. Reagents were Mallinckrodt analytical grade, and solvents were re-distilled in all-glass apparatus before use. Heptafluorobutyric anhydride and amino acid standards were obtained from Pierce Chemical Co. RESULTS AND DISCUSSION The locations of the sampling stations are shown in Fig.1. Stations 36--49 were selected from a larger number of sampling locations, extending from 28°N to 28°S, to illustrate the changes which might be encountered when passing from a region of relatively high temperature and productivity at the

218

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Fig. 1. Sampling locations for suspended particulate matter.

e q u a t o r to the oligotrophic South Pacific Gyre in Austral winter. Stations 103--401 represent coastal water influenced by the California current and the input o f terrigenous material. The depth profile at Station 106 includes a sample '~rom relatively stagnant waters below the sill depth of the San Clemente Basin. Figure 2 shows examples of the gas chromatograms from samples at three depths at Station 106. The increasing unevenness of the baseline for deeper

219

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Fig.2. Gas chromatographic separations of the N-heptafluorobutyryl, O-isobutyl derivatives of amino acids from particulate material obtained from three depths at Station 106 (San Clemente Basin). GC column: 6 m x 2 mm ID glass column filled with 3% OV-101 on Gaschrom Q; carrier gas: N2 at 16 ml/min; injector 210°C, flame ionization detector 280°C; temperature program 80°C isothermal for 5 rain, then 6°C/rain to 250°(]. BL = peaks which also occur in a blank sample without amino acids.

220

samples is a result of higher instrument sensitivity at low amino-acid concentrations. The " b l a n k " peaks are a combination of reagent impurities and septum bleed from the gas chromatograph. For the 1250-m sample, the isoleucine peak, for example, represents approximately 2 nmole of this aminoacid derivative injected into the gas chromatograph. With this sensitivity, it is necessary to filter only 1--2 1 of surface water and 4--6 1 of deep ocean water

TABLE I A m i n o acid c o n c e n t r a t i o n a n d mole % c o m p o s i t i o n in p a r t i c u l a t e m a t t e r f r o m California coastal waters Amino acid residue

Ala

Gly Val Thr Ser a

Leu Ile Pro Hyp b Met

Station 103 Sept. 14, 1 9 7 4

S t a t i o n 201 Sept. 15, 1 9 7 4

lm

lm

18 m

nmole/1

%

nmole/1

%

105 125 65 56 76 8 74 38 53 2 3 25

10.7 12.8 6.6 5.7 7.8 0.8 7.6 3.9 5.4 0.2 0.3 2.6

112 148 45 131 159 10 136 33 68 + 8 58

6.7 8.9 2.7 7.9 9.6 0.6 8.2 2.0 4.1

103 32 136 58 21

10.5 3.3 13.9 5.9 2.1

204 83 282 130 53

12.3 5.0 17.0 7.8 3.2

Lys Tyr PAA (nmole/l) P A A (pg/l)

98O 119

nmole/1

%

nmole/1

%

75 100 41 61 80 2 58 31 34

8.6 11.5 4.7 7.0 9.2 0.2 6.6 3.6 3.9

76 127 42 72 113 1 65 34 39

6.6 11.1 3.7 6.3 9.8 0.1 5.7 3.0 3.4

.}-

0.5 3.5

C

Asp Phe Glu

28 m

1660 211

1 20 2 105 29 160 51 23

0.1 2.3 0.2 12.0 3.3 18.3 5.8 2.6 873 108

__

__

2 24 22 152 35 258 60 27

0.2 2.1 1.9 13.2 3.0 22.5 5.2 2.3 1150 144

P A A - N (pg/l) P O N (pg/l) P A A - N / P O N (%)

14.5 18.8 77

25.1 22.1 114

12.9 13.8 93

16.9 18.7 90

PAA-C (pg/l) POC (pg/l) PAA-C/POC (%)

53.O 199 27

94.2 182 52

47.0 108 44

62.3 177 35

a, b, c, see text. +, p r e s e n t , b u t b e l o w 0.1% o f total. --, n o t d e t e c t e d .

108 393 27

29.9 40.9 73

1996 241

161 77 261 138 52

.

.

8.1 3.9 13.1 6.9 2.6

2.6

8.5 4.1 5.1 0.3

.

.

117 56 240 101 42

.

7.8 3.7 16.0 6.7 2.8

10.6 12.6 6.1 6)* 9)* . 7.6 3.8 4.6 0.1 . 2.5

82.3 518 16

22.4 43.9 51

1500 183

114 57 69 2 . 37

159 189 92 ( 90 (135

.

.

9.2 5.2 12.3 6.9 4.7

20.2 73.0 27

2.1

7.1 4.4 3.9 --

9.4 13.8 5.8 6)* 9)*

5.50 7.30 75

367 45

33.9 19.0 45.1 25.5 17.4

7.8

34.5 50.5 21.4 (22.0 (33.0 . 26.1 16.2 14.4 -.

141 33 163 60 20

67 36 67 3 2 25

124 158 72 74 91

12.4 2.9 14.3 5.3 1.8

5.9 3.2 5.9 0.3 0.2 2.2

10.9 13.9 6.3 6.5 8.0

%

59.0 141 42

16.7 17.1 98

1136 136

nmole/l

10.2 13.4 6.3 6)* 9)*

%

203 268 126 (120 (180 . 169 82 101 6 . 52

nmole/l

nmole/l

%

nmole/l

%

lm

20m

lm

38m

S t a t i o n 401 Sept. 19, 1 9 7 4

S t a t i o n 303 Sept. 16, 1 9 7 4

236 74 290 142 38

262 293 153 138 172 11 160 80 133 5 13 58 10.5 3.3 12.8 6.3 1.7

11.6 13.0 6.8 6.1 7.6 0.5 7.1 3.5 5.9 0.2 0.6 2.6

%

120 587 2O

33.6 45.8 73

2258 271

nmole/l

4m

* Percentages Thr and Ser were estimated at 6% and 9 % respectively, because G C peak integration was incorrect.

PAA-C (/~g/l) P O C (/~g/l) PAA-C/POC (%)

P O N (pg/l) P A A - N / P O N (%)

P A A - N (/ig/l)

Z P A A (/~g/l)

PAA (nmole/l)

Wyr

Asp Phe Glu Lys

C

b Met

Hyp

Gly Val Thr Ser a Leu Ile Pro

Ala

Amino acid residue

T A B L E I, c o n c l u d e d

tO

b~

14.0 42.6 33

PAA-C (pg/1) POC (pg/l) P A A - C / P O C (%)

5.1 10.2 50

1.34 1.5 89

91.6 11.5

8.3 10.9 4.8 3.5 5.5 6.9 3.7 4.3 0.2 2.2 0.3 17.1 0.1 4.1 20.6 4.8 2.6

%

2.6 7.0 37

0.73 1.1 66

50.0 6.0

4.8 8.0 2.8 2.6 5.0 3.7 2.0 2.0 0.2 0.5 0.4 6.5 0.1 1.8 6.1 2.3 1.2

nmole/l

150 m

1.5 4.1 37

0.43 0.7 61

29.2 3.5

2.9 4.4 1.7 1.7 2.5 2.2 1.3 1.2 0.1 0.3 0.5 3.9 0.1 1.0 3.3 1.4 0.7

nmole/1

200 m

* Tyr was estimated at 2.5% because G C peak integration was incorrect. ** Exceptionally high value for c o m p o n e n t c; not used in calculations.

3.88 3.6 108

261.8 31.9

P A A - N (/~g/1) PON (pg/1) P A A - N / P O N (%)

P A A (nmole/1) P A A (pg/l)

7.6 10.0 4.4 3.2 5.0 6.3 3.4 3.9 0.2 2.0 0.3 15.7 0.1 3.8 18.9 4.4 2.4

26.2 34.5 14.6 15.3 19.3 18.1 10.5 10.9 0.5 5.3 -37.2 1.5 8.9 38.3 15.1 5.6

Ala Gly Val Thr Ser Leu Ile Pro Hyp Met c Asp d Phe Glu Lys Tyr

10.0 13.2 5.6 5.8 7.4 6.9 4.0 4.2 0.2 2.0 -14.2 0.6 3.4 14.6 5.8 2.1

nmole/l

nmole/l

%

100 m

1 m

Amino acid residue,

1.9 5.6 34

0.55 0.6 92

36.7 4.4

3.1 6.8 1.7 1.9 3.4 2.3 1.3 1.5 . 0.4 (3.3)** 4.6 0.2 1.3 4.7 2.6 (0.9)*

nmole/1

240 m

36.2 4.2

0.5 0.3 2.5 0.3 1.3 3.9 1.8 0.9

1.8 5.4 33

0.53 0.3 177

.

3.0 8.5 1.7 1.9 4.4 2.5 1.3 1.4

nmole/1

490 m

.

1.1 2.5 44

0.35 0.2 175

24.5 2.8

1.6 6.6 1.0 0.9 3.8 1.7 0.8 0.5 . 0.1 0.1 2.4 + 0.7 3.2 0.5 (0.6)*

nmole/1

740 m

0.8 3.3 24

0.23 0.2 115

15.7 1.8

1.3 3.4 0.7 0.8 1.8 1.0 0.5 0.6 . 0.1 0.1 1.7 0.1 0.5 1.9 0.8 (0.4)*

nmole/1

1000 m

15.1 1.7

0.1 -1.8 -0.4 2.3 0.4 (0.4)*

0.7 3.6 19

0.22 0.2 110

.

1,2 3.4 0.8 0.5 2.0 1.0 0.5 0.3

nmole/1

2000 m

0.6 3.1 19

0.16 0.2 80

11.1 1.3

0.1 -1.7 -0.3 1.7 0.2 (0.3)*

0.8 2.2 0.6 0.4 1.6 0.6 0.3 0.3

nmole/1

3000 m

Vertical d i s t r i b u t i o n o f a m i n o acids~ t o t a l c a r b o n , a n d t o t a l n i t r o g e n in p a r t i c u l a t e m a t t e r f r o m t h e e q u a t o r i a l Pacific Ocean, S t a t i o n 36, July 15--16, 1972

TABLEII bD

223

to obtain sufficient particulate material for a suitable analysis. This greatly simplifies the sampling procedure. Amino acid concentrations were calculated from the gas chromatograms (Siezen and Mague, 1977) and are shown in Tables I--IV. Particulate aminoacid nitrogen (PAA--N)and carbon (PAA--C) concentrations were calculated and compared with total PON and POC as determined independently for separate samples by an C--H--N analyzer (Tables I--IV). Unfortunately, in our hands, arginine, histidine and cysteine/cystine did not reproducibly survive the combination of hydrolysis, ion-exchange chromatography and derivatization, (Siezen and Mague, 1977) and so these amino acids are not quantitatively reported, even though they appear in small amounts on the

T A B L E III A m i n o acid c o n c e n t r a t i o n a n d m o l e - p e r c e n t c o m p o s i t i o n in p a r t i c u l a t e m a t t e r f r o m t h e s u b - t r o p i c a l S o u t h Pacific O c e a n Amino acid residue

S t a t i o n 43 J u l y 22, 1 9 7 2 lm

64m

nmole/1 Ala

Gly Val Thr Set Leu Ile Pro Hyp Met C

Asp Phe Glu Lys

Tyr Z PAA (nmole/l) Z P A A (pg/1)

17.3 20.9 9.3 10.4 14.6 11.7 7.7 7.0 0.5 3.6 1.0 22.8 5.5 24.3 9.7 5.7

S t a t i o n 45 J u l y 23, 1 9 7 2

% 10.1 12.2 5.4 6.0 8.5 6.8 4.5 4.1 0.3 2.1 0.6 13.3 3.2 14.1 5.6 " 3.3

172.0 21.1

86 m

lm

nmole/1

%

nmole/1

%

nmole/1

%

19.5 24.9 10.9 11.6 16.7 13.3 8.8 7.9 0.5 3.9 -24.2 6.1 26.0 11.6 6.2

10.2 13.0 5.7 6.0 8.7 6.9 4.6 4.1 0.3 2.0 -12.6 3.2 13.5 6.0 3.2

9.5 13.7 5.6 6.6 9.8 7.3 4.5 4.9 0.3 2.6 1.0 13.8 3.9 13.3 5.3 2.6

9.1 13.1 5.3 6.3 9.4 7.0 4.3 4.7 0.3 2.5 1.0 13.2 3.7 12.7 5.1 2.5

15.2 25.6 8.5 8.8 15.4 10.7 7.0 5.8 0.4 3.1 0.2 18.0 4.6 20.4 7.5 4.4.

9.8 16.5 5.5 5.7 9.9 6.9 4.5 3.7 0.3 2.0 0.1 11.6 3.0 13.1 4.8 2.8

192.1 23.4

104.7 12.7

155.6 18.5

P A A - N (pg/1) PON (pg/l) P A A - N / P O N (%)

2.54 3.2 79

2.85 3.1 92

1.54 1.8 86

2.28 2.6 88

P A A - C (]~g/1)

9.3 21.2 44

10.3 21.5 48

5.6 16.8 33

8.1 18.2 45

P O C (~gll)

PAA-C/POC (%) c, see t e x t .

93.6 11.3

1.37 1.4 98

5.0 10.0 50

P A A - N (pg]l) PON (pg/1) P A A - N / P O N (%)

P A A - C (~g/1) POC (pg/l) P A A - C / P O C (%)

+, present, b u t b e l o w 0.1% o f total.

P A A (pg/1)

10.8 14.2 3.3 4.1 6.6 7.6 2.0 4.7 0.3 1.4 0.7 8.9 4.2 11.5 4.2 2.4

4.6 12.0 38

1.82 1.6 78

86.9 10.4

12.4 16.3 3.8 4.8 7.6 8.7 2.3 5.4 0.3 1.6 0.8 10.2 4.8 13.2 4.8 2.8 3.4 7.1 2.3 2.0 4.3 3.1 1.8 1.6 + 0.4 0.6 4.5 2.2 5.4 1.9 1.1

2.2 9.8 22

0.61 0.6 102

41.7 5.0

1.0 1.4 10.8 5.3 13.0 4.6 2.6

8.2 17.0 5.5 4.8 10.3 7.4 4.3 3.8

9.3 13.2 5.9 5.3 8.0 7.7 4.8 4.0 0.3 1.5 1.0 10.9 4.1 12.6 4.6 2.7

9.7 13.8 6.2 5.5 8.3 8.0 5.0 4.2 0.3 1.6 1.0 11.4 4.3 13.1 4.8 2.8

%

5.2 11.3 46

1.41 1.2 117

95.9 11.6

nmole/1

10.8 14.6 5.7 5.7 9.0 7.5 4.3 3.7 0.2 1.4 1.1 11.5 4.1 13.0 4.6 2.9

%

10.1 13.7 5.3 5.3 8.4 7.0 4.0 3.5 0.2 1.3 1.0 10.8 3.8 12.2 4.3 2.7

nmole/1

nmole/1

%

nmole/1 %

2m

93 m

lm 200 m

Station 49 J u l y 25, 1 9 7 2

S t a t i o n 47 J u l y 24, 1 9 7 2

~, P A A (nmole/1)

Asp Phe Glu Lys Tyr

C

Gly Val Thr Ser Leu ne Pro Hyp Met

Ala

acid residue

Amino

T A B L E III, c o n c l u d e d

85 m

11.0 13.8 6.1 6.2 9.3 7.6 4.8 4.3 0.4 2.2 0.6 11.5 3.4 11.8 5.2 2.9

10.9 13.6 6.0 6.1 9.2 7.5 4.7 4.3 0.4 2.2 0.6 11.4 3.4 11.7 5.1 2.9

%

5.4 11.7 46

1.49 1.6 96

101.1 12.2

nmole/l

200 m

3.7 5.8 2.3 2.2 3.6 3.2 1.9 1.7 0.1 0.5 1.1 4.6 2.1 4.8 2.3 1.1

2.2 6.4 34

0.61 0.7 91

41.0 5.0

nmole/1

9.0 14.1 5.6 5.4 8.8 7.8 4.6 4.1 0.2 1.2 2.7 11.2 5.1 11.7 5.6 2.7

%

t~

18.3 41.5 44

PAA-C (pg/l) POC (pg/l) P A A - C / P O C (%)

5.40 22.5 24

1.49 2.23 67

100.7 12.0

11.2 17.1 5.9 6.8 6.4 7.7 4.6 4.4 0.2 2.5 8.6 4.1 12.2 5.6 2:9

%

4.04 10.3 39

1.09 0.80 136

74.6 9.0

7.0 10.9 3.7 5.0 5.7 5.7 2.2 4.8 0.5 1.6 8.1 2.5 11.4 3.6 1.9

nmole/1

100 m

1.94 9.3 20

0.52 0.53 98

35.4 4.2

3.9 (5.3)* 1.9 2.1 3.5 2.9 1.7 1.3 + 0.6 3.5 1.2 4.5 1.8 1.2

nmole/1

250 m

2.03 8.1 25

0.56 0.95 59

37.8 4.6

2.6 6.2 1.6 2.0 3.9 3.0 1.6 1.8 + 0.6 4.2 1.2 5.8 2.2 1.1

nmole/1

500 m

1.60 8.3 19

0.45 0.46 98

30.8 3.6

2.6 6.6 1.4 1.8 3.1 2.5 1.4 1.2 + 0.5 2.8 0.9 3.8 1.4 0.8

nmole/1

750 m

1.51 7.0 21

0.43 0.46 93

28.6 3.4

1.8 5.3 1.4 1.4 3.0 2.3 1.3 1.0 -0.4 3.2 1.0 4.0 1.8 0.7

nmole/1

1000 m

* Gly was e s t i m a t e d , 15% at 2 5 0 m a n d 20% a t 1800 m, because GC p e a k integration was incorrect.

5.00 5.58 90

399.0 41.3

P A A - N (~g]l) P O N (gg/1) P A A - N / P O N (%)

P A A (nmole/1) P A A (rag/l)

11.3 17.2 5.9 6.8 6.4 7.8 4.6 4.4 0.2 2.5 8.7 4.1 12.3 5.6 2.9

34.0 45.9 18.1 24.0 22.1 23.4 12.6 17.8 0.9 9.3 39.0 13.5 51.4 18.2 8.8

Ala Gly Val Thr Ser Leu lie Pro Hyp Met Asp Phe Glu Lys Tyr

10.0 13.5 5.3 7.1 6.5 6.9 3.7 5.3 0.3 2.7 11.5 4.0 15.2 5.4 2.6

nmole/1

nmole/l

%

55 m

1 m

Amino acid residue

1.52 6.3 24

0.44 0.34 129

29.8 3.5

2.2 7.0 1.5 1.3 2.9 2.2 1.0 1.1 + 0.6 2.7 1.0 3.9 1.6 0.8

nmole/1

1250 m

1.50 5.5 27

0.43 0.47 91

28.9 3.4

2.1 5.2 1.4 1.3 3.3 2.5 1.1 1.1 -0.6 3.1 0.8 4.1 1.7 0.6

nmole/1

1500 m

1.10 9.4 12

0.32 0.39 82

21.9 2.5

2.4 (4.4)* 1.1 1.1 2.9 1.5 0.8 0.8 -0.4 2.2 0.7 2.1 1.0 0.5

nmole/1

1800 m

Vertical d i s t r i b u t i o n o f a m i n o acids, t o t a l c a r b o n , a n d t o t a l n i t r o g e n in p a r t i c u l a t e m a t t e r f r o m o f f s h o r e California waters, S t a t i o n 106, Sept. 18, 1 9 7 4

TABLE IV

b~

226 chromatograms. Generally, histidine and cysteine/cystine do not a m o u n t to more than 1--2% of the particulate amino acids (PAA), but arginine may comprise up to 10% of total amino acid residues (Degens, 1970). Therefore, the total PAA and PAA-C concentration will be slightly higher {5--10%) than listed in the tables, and the PAA-N may be significantly higher {20--40%) than listed, since arginine contains four N-atoms. Small amounts of unidentified components (called a, b, c and d in the Tables), presumably u n c o m m o n amino acids, are found mainly in the euphotic zone, especially in the presence of a phytoplankton bloom (Siezen and Mague, 1977}. In all calculations, these components were assumed to have a molecular weight of 130, one N-atom, six C-atoms and a molar response of 1.0 relative to the internal standard. These assumptions are based upon the order of elution of these unidentified components, relative to the c o m m o n amino acids, during gas chromatography. Absolute concentrations of PAA were highest in the nearshore euphotic zone {Table I). The maximum was usually at the surface except at shallow stations (201 and 401) where the influence of suspended sediment in deeper samples is likely. At Station 401, in the cooling water discharge plume of the San Onofre nuclear generating plant, the high subsurface concentrations of PAA may b e due both to sediment disruption and to the discharge of flagments of larger organisms which would not normally be sampled if intact and alive. For the open-ocean stations, the concentration of PAA was highest in surface waters at the equator where increased p h y t o p l a n k t o n growth is possible due to higher temperatures and mixing of nutrients from deeper waters (Table II). As the colder, nutrient-depleted waters of the South Pacific gyre are approached {Stations 43--49; Table III) the total PAA concentration decreases. However, the concentration is generally higher in the 60--90 m zone than at the surface, reflecting the relatively deep p h y t o p l a n k t o n maxim u m found in oligotrophic areas. It is interesting to note that at the 200 m depth at stations 47 and 49, PAA are more abundant than at the corresponding depth at the more productive equatorial Station 36. This may be the result of a more rapid and intense production and turnover of material at the latter location. The two depth profiles {Tables II and IV) show that PAA decrease rapdily with depth to a b o u t 200 m, levelling off at 20--30 nmoles/1 in the San Clemente Basin and 10--20 nmoles/1 in Central Pacific equatorial water. Changes below 1000 m depth are relatively small and generally support the conclusions of Menzel and Ryther {1970) that the majority c f biochemical transformations of organic matter in the oceans takes place above a depth of 300 m. It is clear that the overall amino-acid composition of particulate matter is very similar in all waters investigated {Table V). Even the highest standard deviation found (for glycine) was no more than 4.1 mole%, while the highest and lowest coefficients of variability observed were for methionine and leucine,

227 TABLE V A m i n o acid c o m p o s i t i o n (in m o l e %) o f s u s p e n d e d p a r t i c u l a t e m a t e r i a l averaged s e p a r a t e l y for s h a l l o w coastal a n d o c e a n i c s a m p l i n g areas a n d for all samples at all s t a t i o n s Amino acid residue

Ala Gly Val Thr Set Leu Ile Pro Met Asp Phe Glu Lys Tyr Others

Above 100 meters coastal

All d e p t h s oceanic

all

all samples

% Av

S.D.

V

% Av

S.D.

V

%Av

S.D.

V

%Av

S.D.

V

9.7 12.9 5.5 6.7 8.1 7.2 3.6 4.7 2.5 10.6 3.8 15.2 6.2 2.7 0.7

1.7 2.0 1.3 0.7 1.3 0.9 0.7 0.9 0.4 1.9 0.8 3.1 0.8 0.8 --

18 16 24 10 16 13 19 19 16 18 21 20 13 30 --

10.3 14.0 5.5 5.8 8.7 7.4 4.2 4.3 1.9 12,2 3.7 13.2 5.2 2.8 0.9

0.9 1.5 0.7 0.4 0.8 0.6 0.8 0.5 0.3 1.2 0.6 0.8 0.5 0.4 --

9 11 13 7 9 8 19 12 16 10 16 6 10 14 --

10.0 13.4 5.5 6.2 8.4 7.3 3.9 4.5 2.3 11.3 3.7 14.3 5.7 2.7 0.8

1.4 1.9 1.0 0.7 1.1 0.8 0.8 0.7 0.5 1.8 0.7 2.5 0.8 0.6 --

14 14 18 11 13 11 21 16 22 16 19 17 14 22 --

9.2 15.9 5.2 5.5 9.6 7.3 3.9 4.2 1.8 11.3 3.6 13.9 5.3 2.7 0.8

1.6 4.1 0.8 1.0 2.2 0.8 0.7 0.9 0.7 2.0 0.7 2.5 1.2 0.5 --

17 26 15 18 23 11 18 21 39 18 19 18 23 19 --

S.D., s t a n d a r d deviation. V, c o e f f i c i e n t o f variation, %. --, i n s u f f i c i e n t data for calculation.

respectively. S o m e slight, but significant, trends can be detected, however. In Fig.3, the PAA content of POM at Stations 36 and 106 is compared on the basis of mole % composition. The relative amounts of glycine and serine increase at greater depths, and together with aspartic acid and glutamic acid t h e y are the most abundant particulate amino acids in deep water. Hydroxyproline decreased with depth to unmeasurably low values at both stations and methionine and lysine decreased slightly in the deeper samples only at Station 36. The absence of certain amino acid residues is interesting. It is reasonable to suspect that particulate material below the euphotic zone may contain a relatively large proportion of algal cellular debris in which the cell wall protein would be the most resistant to attack. The u n c o m m o n amino acids, a, ediaminopimelic acid, 3,4-dihydroxyproline and e-N-trimethyl-L-hydroxylysine have been reported as constituents of certain algal cell wail proteins (HolmHansen et al., 1965; Nakajima and Volcani, 1969, 1970). Our chromatographic m e t h o d (Siezen and Mague, 1977) can resolve and measure these first two compounds; however, neither of these amino acids were detected in our samples. Also, the fact that the more c o m m o n cell-wall constituent, hydroxyproline, decreased to unmeasurable levels with depth suggests that simple loss

228 4 LO> f_~(/) u -- D_:g =E ,~[ ~ LO.Ji-

Im

20

IOI

~

0

~o?

IOO~ ~

~

'°~

55m

0

2°~1~ 20

~

150m

L:'o~

200m

~I~N

~o~

o

o,o~ z°t

~

o

~ 121

H

r'~

240m

~~

750m

7 4 0 rn

~tR

I~o

IOOOm

':,o~ ~

125orn

20

--

~~~

,o-

~o~ 'i? Io

o

2o~

0

500 m

~ _

v

25Om

~~N v

2OOOm

~

~

HI Hi

~o] ~

,5oo~

u

zo~

r8OOm

SOUTHERN CALIFORNIA CENTRAL PACIFIC EOUATORIAL WATER COASTAL WATER STATION 36 STATION IO6 Fig.& Depth profiles of the relative amino acid composition, in mole % (residues per 100), of suspended particulate matter.

229

of cellular contents is insufficient for explaining the decay of phytoplankton debris. On the other hand, the relative increase with depth of serine and glycine which we observed may be due in part to the persistence of a protein-silica complex in diatom cell walls, enriched in serine, threonine and glycine, which has been proposed by Hecky et al. (1973). However, there is no significant increase in threonine in our samples. We also looked specifically for, but failed to detect, ornithine (reported by Degens, 1970, and cited as evidence for degeneration of plankton material) or the 2-amino butyric acids which are present in some diatoms (Chuecas and Riley, 1969). These amino acids, if present, would have been measured by the method used. Nonetheless, the amino-acid composition of POM which we have found in surface water shows reasonable similarity to the average amino acid composition of the several marine diatom species described by Chuecas and Riley (1969) and is nearly identical to the composition of "plankton" reported by Degens (1970; table I). The calculated nitrogen content of the total particulate amino acid pool (PAA--N) is sufficient, in many cases, to account for all the PON as measured by high-temperature combustion (Tables I--IV), indicating that the conversion of amino acids to refractile, heterocyclic compounds in deep water is relatively unimportant, at least for material retained on glass-fiber filters. However, agreement was not uniform and PAA-N ranged from 51 to 177% of PON for all stations sampled. (The high percentage values occur in deeper samples at station 36 (Table II) and almost certainly reflect errors in PON determination by CHN analyzer at these very low N values which were close to the limit of detection of the instrument. The amino acid analyses, on the other hand, are more reliable since sufficient material was available for the gas chromatograpy). The low percentage values were found at Station 303 (Table I) near the Hyperion sewer outfall at Santa Monica. This may indicate that sewage introduces substantial amounts of nitrogenous organic compounds other than amino acids into seawater. The contribution of particulate amino-acid carbon (PAA-C) to the total POC was much less than for nitrogen and ranged from about 20 to 50%. The highest values were found in near-surface samples from California coastal waters, where phytoplankton growth was abundant, and in the upper 100 m in the Pacific Ocean. PAA-C is less than 27% of POC below 1000 m for both coastal and open-Qcean water (Tables II and IV), with a minimum mean of 20% in both cases. These data suggest that the proteinaceous content of plankton is relatively more labile than other carbon-rich constituents and is preferentially degraded during sinking to great depths (see also Gordon, 1970). Handa et al. (1972) demonstrated the presence of refractory polysaccharides in POM from the Pacific Ocean, and the presence of these compounds in our samples may be responsible for the observed decrease with depth in PAA-C/ POC. An exception to this general pattern was, again, Station 303 (Table I) where sewage probably contributes considerable non-protein POC (possibly of cellulose origin).

230

The total amount of PAA, expressed in pg/1, is a more accurate measure o f particulate protein than are determinations based upon reactions of terminal or side groups (fluorescamine, Folin's reagent), although, admittedly, total amino acid analyses are rather laborious for routine protein determinations. Our "protein equivalent" values range from 11 to 32 pg/l in the upper 100 m of the central Pacific Ocean, from 1.3 to 6.0 pg/1 below 100 m, and from 45 to 270 ~g/1 in California coastal water. Packard and Dortch (1975) measured fluorescamine-positive material as "~=casein equivalents" and found (our re-calculation) 13--60 pg/1 (occasionally up to 140 pg/1) in the upper 100 m of the central Atlantic Ocean and 40--300 pg/1 in Spanish Sahara coastal waters. This indicates that an indirect measurement is probably valid for estimating the protein content of POM. However, a knowledge of the total amino acid composition provides greater insight into the nature of POM and the transforming processes which modify it.

ACKNOWLEDGEMENTS

We thank Dr. O. Holm-Hansen and other members of the F o o d Chain Research Group for providing facilities and financial assistance for this research and F. C. Mague for performing the analyses for POC and PON. Support was provided by National Science Foundation Grants, GA-36511, GV-27110 and DES-20956, U.S. Atomic Energy Commission Contract No. AT {11-1) GEN 10, P.A. 20 and U.S. Energy Research and Development Administration Contract E (11-1) GEN 10, P.A. 20. NATO-fellowship N84-117 was awarded to R. J. Siezen by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).

REFERENCES Chester, R. and Stoner, J. H., 1974. The distribution of particulate organic carbon and nitrogen in some surface waters of the World Ocean. Mar. Chem., 2: 263--275. Chuecas, L. and Riley, J. P., 1969. The component combined amino acids of some marine diatoms. J. Mar. Biol. Assoc. U.K., 49: 117--120. Degens, E. T., 1970. Molecular nature of nitrogenous compounds in sea water and recent marine sediments. In: D. W. Hood (Editor), Organic Matter in Natural Waters. University of Alaska, Fairbanks, pp.77--106. Degens, E. T., Reuter, J. H. and Shaw, K. N. F., 1964. Biochemical compounds in offshore California sediments and sea waters. Geochim. Cosmochim. Acta, 2 8 : 4 5 - - 6 6 . Gordon, D. C., 1970. Some studies on the distribution and composition of particulate organic carbon in the North Atlantic Ocean. Deep-Sea Res., 17: 233--243. Handa, N., 1970. Dissolved and particulate carbohydrates. In: D. W. Hood (Editor), Organic Matter in Natural Waters. University of Alaska, Fairbanks, pp.129--152. Handa, N., Yanagi, K. and Matsunage, K., 1972. Distribution of detrital materials in the Western Pacific Ocean and their biological nature. Mere. Ist. Ital. Idrobiol., 29 (Suppl.): 53--71.

231 Hecky, R. E., Mopper, K., Kilham, P. and Degens, E. T., 1973. The amino acid and sugar composition of diatom cell-walls. Mar. Biol., 19: 323--331. Holm-Hansen, O., 1972. The distribution and chemical composition of particulate material in marine and fresh waters. Mem. Ist. Ital. Idrobiol., 29 (Suppl.): 37--51. Holm-Hansen, O., Prasad, R. and Lewin, R. A., 1965. Occurrence of (~, e-diaminopimelic acid in algae and flexibacteria. Phycologia, 5: 1--14. Holm-Hansen, O., Strickland, J. D. H. and Williams, P. M., 1966. A detailed analysis of biologically important substances in a profile off Southern California. Limnol. Oceanogr., i i : 548--561. MacKenzie, S. L. and Tenaschuk, D., 1974. Gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters of amino acids. J. Chromatogr., 97: 19--24. Menzel, D. W. and Ryther, J. H., 1970. Distribution and cycling of organic matter in the oceans. In: D. W. Hood (Editor), Organic Matter in Natural Waters~ University of Alaska, Fairbanks, pp. 31--54. Nakajima, T. and Volcani, B. E., 1969. 3,4-dihydroxyproline: a new amino acid in diatom cell walls. Science, 164: 1400--1401. Nakajima, T. and Volcani, B. E., 1970. e-N-trimethyl-L-6-hydroxylysinephosphate and its nonphosphorylated compound in diatom cell walls. Biochem. Biophys. Res. Comm., 39: 28--33. Packard, T. T. and Dortch, Q., 1975. Particulate protein-nitrogen in North Atlantic surface waters. Mar. Biol., 33: 347--354. Parsons, T. R. and Strickland, J. D. H., 1962. Oceanic detritus. Science, 136: 313--314. Sharp, J. H., 1974. Improved analysis for "particulate" organic carbon and nitrogen from seawater. Limnol. Oceanogr., 19: 984--989. Siezen, R. J. and Mague, T. H., 1977. Gas-liquid chromatography of the N-heptafluorobutyryl isobutyl esters of fifty biologically interesting amino acids. J. Chromatogr., 130: 151--160.