Amino acid compositions of the egg-yolk lipoproteins, and a statistical comparison of their amino acid ratios

98

BIOCHIMICA ET BIOPHYSICA ACTA

AMINO ACID COMPOSITIONS

OF THE

EGG-YOLK

AND A STATISTICAL COMPARISON THEIR

LIPOPROTEINS, OF

AMINO ACID RATIOS*

W. H. COOK, R. W. BURLEY, W. G. MARTIN AND J. ~v. HOPKINS Division of Applied Biology, National Research Council, Ottawa (Canada)

(Received November 27th, I961) (Revised manuscript received March 6th, t962 )

SUMMARY The amino acid compositions of ~- and fl-vitellin and vitellenin, representing, respectively, the protein parts of ~- and fl-lipovitellin and the low-density fraction of egg yolk, have been determined. As these proteins were quite similar in their amino acid compositions, a statistical examination of the disproportionalities in their amino acid ratios formed the basis of comparison. In addition to a difference in phosphorus content, viteUenin differs from both vitellins in its content of IO amino acids. The lipovitellins cannot therefore be converted to the low-density fraction containing vitellenin, or vice versa, by a mere loss or gain of lipid. The 2 vitellins differ only in their content of phosphorus and histidine.

INTRODUCTION Hen's egg yolk contains three physically-distinct lipoproteins 1, a- and fl-lipovitellin, each of 22 ~o lipid content, and a L D F of 89 % lipid content. The separation, purification and characterization of these lipoproteins depend primarily on differences in their physical properties 2-4. Apart from the higher lipid content of L D F , t h e y are all quite similar in chemical composition. A small difference in the protein-phosphorus content of a- and fl-lipovitellin is the only other n o t e w o r t h y difference in composition t h a t has been demonstrated ~, 3 b u t the physical behavior of these lipoproteins 5 suggests t h a t their protein moieties m a y differ in other respects. Following lipid extraction all three protein residues are insoluble in other than strong acids and alkalies 4 and, although t h e y have been termed ~- and fl-vitellin and vitellenin**, this is only to distinguish t h e m from the corresponding parent lipoproteins. All three of these proteins have the same m a j o r N-terminal amino acidsT: the amino acid compositions of ~- and ~-vitellin appear to be the same ~ and t h a t of vitellenin has not been reported. Recent refinements 3 in the procedures for preparing the parent lipoproteins have reduced the phosphorus content of both vitellins, indicating t h a t earlier preparations 2 Abbreviation: LDF, low-density fraction. * Contribution from Division of Applied Biology, National Research Council, Ottawa (Canada). Issued as N.R.C. No. 6827. ** F~VOLD AND LAUSTEN6 obtained "lipovitellenin", a lipoprotein with 4o% lipid, by centrifuging diluted egg yolk and ether extracting the supernatant solution containing LDF. The lipid-free protein, common to LDF and lipovitellenin, is referred to as "vitellenin". Biochim. Biophvs. Acta. 60 (~c~62) 08 lo 3

AMINO ACIDS AND THEIR RATIOS IN YOLK LIPOPROTEINS

99

m a y have been contaminated with phosvitin. The protein-phosphorus content of L D F prepared b y these new procedures has not been reported. The present study was undertaken to determine the amino acid contents of all three lipoproteins prepared b y the improved procedures. These results were also used to ascertain if there were any detectable differences in the composition of their polypeptide chains. To minimize the effect of any non-protein moieties or contaminants that might be present in these protein residues, a statistical examination of the disproportionalities in their amino acid ratios was used as a basis for comparison. METHODS

The a- and fl-lipovitellins were separated and purified b y the method of BURLEY AND COOKs. Briefly, the sedimenting granules were collected b y centrifuging diluted yolk, then they were washed, dissolved, and the solutions centrifuged to remove the residual LDF. Phosvitin was then removed from this solution by chromatography on a resin column, and the a- and fl-lipovitellins separated b y passage through a liydroxyapatite column. The L D F was prepared from the supernatant solution from the preparation of the granules instead of from solutions of whole yolk in sodium chloride solutions, as described previously 4. Since the granules contain the phosvitin, this modification prevented the interaction of phosvitin with LDF, and yielded preparations having the lowest protein-phosphorus content t h a t has been observed. Following this initial separation the L D F was redispersed in sodium chloride solutions and recentrifuged several times to remove any residual livetin. All three lipoproteins were delipidated b y the BLIGH AND DYER s method and the L D F was purified further b y washing with ethanol to ensure complete removal of lipid. The proteins were freed of solvent i n vacuo and stored over a dehydrating agent. Moisture content was determined b y drying to constant weight at lO5 ° . For amino acid analyses small portions of the proteins of known moisture content were hydrolysed with 6 N hydrochloric acid for 24 h at io5 °, unless otherwise stated, in tubes sealed under vacuum. A Spinco amino acid analyser, model MS, was used for most analyses. Cystine plus cysteine was estimated as cysteic acid on separate samples of protein that had been oxidized at o ° with performic acid 9. Tryptophane was estimated from the tyrosine/tryptophane ratio obtained b y ultraviolet absorption TM and the tyrosine content. Destruction of serine during hydrolysis was estimated b y hydrolysing samples for 24, 48 and 72 h and extrapolating to zero time. The correction to a 24-h hydrolysate ranged from about 5 % for vitellenin to about 15 % for ~-vitellin. Methods for determining phosphorus, sulphur, and nitrogen have already been described ~. RESULTS

The results in Table I represent the average compositions of two independent preparations of vitellenin and three each of a- and fl-vitellin. For comparison the earlier results of BERNARDI AND COOK2 expressed in the same units are included. The two vitellins are quite similar but vitellenin appears to differ from them in its content of some amino acids. Biochim. Biophys. Acta, 60 (1962) 9 8 - I o 3

I00

W. H. COOK, R. W. BURLEY, W. G. MARTIN, J. W. HOPKINS

The earlier preparations of both vitellins 2 had a higher content of phosphorus, nitrogen, and most amino acids than the present proteins. While the higher phosphorus content can be attributed to phosvitin a, the higher nitrogen content is less easily explained. If the amino acid contents of these earlier preparations are adjusted to the nitrogen content of the present samples, thirteen of the eighteen amino acids are in good agreement with the present results. Of the five remaining acids, the difference in the tryptophane estimates m a y be experimental error, and the presence of phosvitin could account for the higher serine content. TABLE I SUMMARY OF AMINO ACID ANALYSIS A m i n o acids in m m o l e s / g d r y p r o t e i n . A mino acid Asp Thr Ser Glu Pro Gly Ala Val Met Ileu Leu Tyr Phe Lys His Arg Try Cys/2 N% P % S o/ /o

Vitellenin (r) 0.80 0.40 o.51.* o.8o o.26 o.36 o.54 o.49 o. 15 o.47 o.79 o.26 o.32 0.6o o.II o.36 o,13"** 0.04 14. 5 o.13 0.87

~-Vitellin (2) 0.60 0.35 o.54"* o.82 o.43 o.38 0.63 o.56 o. 19 o.45 o.7 ° o.23 o.27 o.5o o.19 0.47 o.13"** o.12 15. 7 0.53 1.19

fl-Vitdlin (,3) 0.67 0.36 o.51"* o.86 o.44 o.38 o.61 o.61 o.21 o.49 o.73 o.25 o.25 o.5 r o.15 o.52 o.13"** o.13 15.8 0.3 ° 1.3o

a-Vitellgn* (4) 0.85 0.47 o.8o i.o2 o.48 o.43 o.7o o.55 o.23 o.5o o.82 o.3o 0.29 o.5o o.19 o.48 o.o8 o.16 17.2 1.2o 1.44

fl-Vitellin* (5) 0.83 0.50 o.82 I.O 5 o.48 o.42 o.68 0.58 o.24 o.55 o.8o o.28 o.29 o.53 o.18 o.5o o.o 7 o.18 17.2 0.45 1.2o

* F r o m BERNARDI AND COOK2. ** Corrected for d e s t r u c t i o n d u r i n g h y d r o l y s i s . * ** F r o m T r y / T y r r a t i o a n d T y r content.

Considering the present preparations, estimates of the tryptophane and halfcystine contents are subject to larger errors than the other amino acids, because the former includes the errors of two measurements and the latter will be affected by any phosphoserine that m a y survive hydrolysis, since it coincides chromatographically with cysteic acid, The methionine and half-cystine content of all three proteins parallels their sulphur content but does not account for more than 84 % of the amount present, a difference that m a y fall within the error limits. The over-all experimental errors and the effect of the lower nitrogen content of viteUenin must be considered in assessing the observed differences in composition of the three proteins. Replicate analyses on the same preparation showed that the analytical error of single determinations was about 4.5 % of their mean for each amino acid. The factor limiting reproducibility was, however, the difference between independent preparations of the same protein. I t appears that the main source of B i o c h i m . B i o p h y s . ,4cta, 60 (1962) 9 8 - 1 o 3

AMINO ACIDS AND THEIR RATIOS IN YOLK LIPOPROTEINS

IOI

this variability lies in the non-protein constituents since the standard error of the nitrogen content of individual preparations was 4- 0.53 %. Attempts to distinguish these three proteins by their peptide patterns 11 have so far been unsuccessful. Digestion with trypsin and chymotrypsin yielded relatively few peptides and several combinations of chromatography and high-voltage paper electrophoresis failed to resolve these sufficiently to demonstrate any differences between the proteins. The amino acid ratios of these proteins were, however, examined b y statistical methods for evidence of differences in the composition of their polypeptide chains. As the ratio of one amino acid to another is the same in identical proteins, a variable amount of non-protein constituents is of little consequence if the amino acid ratios within each protein are used as the criterion. Statistical examination was based on this criterion and is similar in principle to that used by BLOCK12, although the present procedure tests the actual amino acid ratio directly. The statistical treatment consisted of an analysis of variance is of the logarithm of the amount (mmoles × Io) of each of fifteen amino acids. Amino acids prone to partial destruction during hydrolysis, e.g., serine, or determined independently, e.g., cystine, were omitted from this comparison, and those subjected to statistical treatment are indicated in Fig. I. This logarithmic transformation was adopted for two reasons. Firstly, as proportionalities are additive on a logarithmic scale, the differences in log concentration of any number of amino acids in each of two distinct proteins or preparations will be constant, regardless of total nitrogen content, if the ratios of their concentrations are constant. Secondly, as the maximal (aspartic) and nadnimal (histidine) amounts of the statistically-compared amino acids for vitellenin differed 1.0

--~-\

0.9 ~

t tTt

O r~

t x //3-VITELLIN

0.7

a-VITELLIN

~ x~-~-O:~,~

,~m

o


0.5

t

I

t

LDF

PROTEIN

x

X~

°

~ IE

0.4

I~

o.3

~

0.2

"

.\

0.1

\ x I I I I I I I I I I I I I f I GLU LEU ASP ALA VAL LY$ i R 6 ILEU PRO 6LY ?HR PHI TYR MI[T Hill

AMINO ACIDS

Fig. I. C o n t e n t of fifteen a m i n o acids in t h e t h r e e e g g - y o l k l i p o p r o t e i n s . S t a r r e d p o i n t s i n d i c a t e a m i n o a c i d s p r e s e n t in s i g n i f i c a n t l y h i g h e r or lower p r o p o r t i o n s t h a n in t h e o t h e r proteins.

Biochim. Biophys. Acta, 60 ~i962 ) 98-1o 3

102

W . H . COOK, R. W. BURLEY, W. G. MARTIN, J. W. HOPKINS

by a factor of 7, this transformation might also tend to "normalize" the scale of independent-preparation discrepancies, making the aggregate estimated error variance reasonably applicablO 3 to the result for each individual acid. TABLE II ANALYSIS OF VARIANCE OF AMINO ACID CONTENT TRANSFORMED TO LOG m m o l e s / g PROTEIN X IO Degreesfreedom

Mean square

A

Average difference b e t w e e n acids for all proteins

14

o.3754o*

B

Aggregate difference between proteins

2

o.oi34o*

C

Aggregate difference between p r e p a r a t i o n s same protein

5

o.oo966*

D

Discrepancies acids x p r o t e i n s (disproportionalities in a m i n o acids) Between c¢- and fl-vitellin Between vitellenin and b o t h vitellins

14 14

0.00184** 0.01586*

d

Discrepancies acids x p r e p a r a t i o n s same protein (estimate of r a n d o m error)

70

V=o.ooo89

* Exceeds o. 1 % significance-level. ** Exceeds 5 % significance-level.

A s u m m a r y of the results of the variance analysis appears in Table II. Some of the quantities, while significant, are of no practical concern. Thus the amino acids (A) are known to be present in different amounts and the mean square between proteins (B) is comparable with that for different preparations of the same protein (C). However, the statistical significance of the variance ratio, Mean square (log acid × log protein) Mean s q u a r e (log acid x log p r e p a r a t i o n s a m e protein)

(i.e., D/d) shows that there is an over-all disproportionality in the amino acids of the three proteins and indicates that they differ in composition. To determine which of the fifteen amino acids contributed significantly to this aggregate disproportionality, the value of D for each amino acid was compared with its standard error computed from d in Table II. The results are summarized in Fig. i for the three proteins (averaged over all preparations and adjusted to the same nitrogen content to facilitate comparison). The amino acids are arranged in decreasing order of quantity; the slope of the connecting lines reflects the difference A (Table II) and the aggregate distance between the lines represents the aggregate difference (B). The individual amino acids showing significant disproportionalities (D) are indicated with asterisks. The lower histidine content of fl-vitellin compared with a-vitellin is the only statistically-significant difference in amino acid composition. No significant differences were found between the a- and fl-vitellins of BERNARDI AND COOK2 when examined by this procedure. Vitellenin differs substantially from the mean of the two vitellins, however, since of the fifteen amino acids studied, six are present in significantly greater, and four in significantly lesser, amounts than in the vitellins. DISCUSSION

These analyses of the protein residues from a- and fl-lipovitellin and L D F (vitellenin), prepared b y improved procedures, provide the best available estimate of their compoBiochim. Bi ophy s . Acta, 60 (I962) 98 lO3

AMINO ACIDS AND THEIR RATIOS IN YOLK LIPOPROTEINS

lO3

sition. Differences in the phosphorus content of a- and fl-vitellin ~,3 have been confirmed, and vitellenin shown to have the lowest phosphorus, sulphur, and nitrogen content. Vitellenin is known to be a glycoprotein 14 and might be expected to have a lower nitrogen content, but the low phosphorus content appears to distinguish it from both vitellins. The present amino acid analyses do permit a rigorous comparison based on the composition of the polypeptide parts of the three proteins. By using as criteria the disproportionalities in the amino acid ratios found in the three proteins, the comparisons are rendered independent of the amounts of all other nitrogenous or nonnitrogenous components that m a y have been present either as contaminants or as part of the parent protein. Replicate preparations of each protein provided a basis for testing the significance of the observed differences b y statistical procedures. A lower proportion of histidine in fl-vitellin was the only statistically-significant difference in the amino acid compositions of the two vitellins but a small difference in one amino acid must be interpreted with caution. Since the parent a- and fl-lipovitellins exhibit different dissociation properties 15 that appear to be unrelated to their protein-phosphorus contents 5, this small difference in amino acid composition m a y be genuine. Vitellenin is clearly different from both vitellins. It has a lower phosphorus content and, indeed, m a y not be a phosphoprotein in the usual sense, since values as low as 0.06 % phosphorus have been observed, and higher values m a y arise from residual phospholipid resulting from oxidation and reduced solubility. Vitellenin also differs from the other two proteins in that ten of the fifteen amino acids studied are present in significantly-different proportions. The previously reported ~ small but consistent differences between the N-terminal residues of vitellenin and those of the vitellins m a y prove to be another distinguishing feature. As L D F and the two lipovitellins have different protein moieties, they cannot be interconverted merely by a loss or gain of lipid. ACKNOWLEDGEMENTS

The authors wish to thank Mr. J. GIROUX, Mr. J. L. LABELLE, and Mr. A. E. CASTAGNE for technical assistance in the preparation and analyses. One of us (R.W.B.) was a Postdoctorate Fellow, 1959-1961. REFERENCES W. H. CooK, Nature, 19o (1961) 1173. G. BERNARDI AND W. H. COOK, Biochim. Biophys. Acta, 44 (196o) 96. R_. W. BURLEY AND W. S . COOK, Can. J. Biochem. and Physiol., 39 (1961) 1295. W. G. MARTIN, K. J. TURNER AND W. H. COOK, Can. J. Biochem. and Physiol., 37 (1959) 1197. R. W. BURLEY AND W. H. COOK, Can. J. Biochem. and Physiol., 4 ° (1962) 373. H. L. FEVOLD AND A. LAUSTEN, Arch. Biochem. Biophys., I I (1946) I. 7 j . M. NEELIN AND W. H. COOK, Can. J. Biochem. and Physiol., 39 (1961) lO75. 8 E. G. BLIGH AND W. J. DYER, Can. J. Biochem. and Physiol., 37 (1959) 911. 9 C. H. \V. HIRS, J. Biol. Chem., 219 (1956) 611. 10 W. L. BENCZE AND K. SCHMID, Anal. Chem., 29 /I957) 1193. 11 M. RODBELL AND D. S. FREDRICKSON, J. Biol. Chem., 234 (1959) 562. 13 R. J. BLOCK, Ann. N . Y . Aead. Sci., 94 (1961) 31. 13 C. A. BENNETT AND N. L. FRANKLIN, Statistical A nalysi~ in Chemistry and the Chemical Industry, W i l e y , N e w Y o r k , 1954, p. 319. 14 S. ABRAHAM, L. A. HILLYARD AND I. L. CHAIKOFF, Arch. Biochem. Biophys., 89 (196o) 7415 1~. W. BURLEY AND W. H. COOK, Can. J. Biochem. and Physiol., 4 ° (1962) 363. 1 2 3 4 5

Biochim. Biophys. Acta, 60 (1962) 9 8 - 1 o 3