Ultraviolet Spectrophotometry of Serum Proteins R. D. STRICKLAND,
P. A. MACK, T. R. PODLESKI, and W. A. CHILDS
Research Division, Veterans Administration Hospital, Albuquerque, N. M. This investigation was undertaken to establish the characteristic absorption spectra of the protein fractions separated from blood serum b y electrophoresis, to evaluate ultraviolet absorbance as a method of estimating serum proteins, and to determine the tyrosine and tryptophan composition of these proteins. This was accomplished for 1 1 protein fractions from each of 12 normal sera. The variations in tyrosine and tryptophan content among the various protein fractions as well as the variations between corresponding fractions from different sera are so wide as to render dangerous the uncritical acceptance of any ultraviolet method for quantitating proteins. Evidence is offered to show that the albumin fraction of serum from women is richer in tyrosine and tryptophan than that from men. HE absorption spectra of serum T p r o t e i n s characteristically have maxima in the region of 290 nip and minima in the region of 270 mp; beyond 270 mp to the limits of detection by ultraviolet spectrophotometry, absorbance increases progressively as shorter wave lengths are scanned. The maximum a t 290 mp is due to the aromatic amino acids. Cysteine, cystine, arginine, the aromatic acids, and the peptide linkage all contribute significantly to absorbance a t wave lengths shorter than 270 mp (1). The strong absorbance of ultraviolet light by proteins offers an attractive method for measuring proteins in minute quantities. If it could be shown that the different proteins of serum have equivalent ultraviolet absorbances, the simplicity and sensitivity of this method would make it excellent for measuring electrophoretically separated serum protein fractions. Holiday (1, 6) has dtwribed a method for estimating tyrosine. and tryptophan in intact proteins by ultraviolet spectrophotometry. Determinations of tyrosine and tryptophan by this method have not been reported for protein fractions separated from serum by electrophoresis in agar gel. This investigation attempted to establish the characteristic absorption spectra of protein fractions electrophoretically separated from blood serum, to evaluate ultraviolet absorbance for estimating serum proteins, and
to determine their tyrosine and tryptophan content. APPARATUS
Beckman spectrophotometers of the
DU and DK series were used alternatively in making these measurements. Matched silica cuvettes with 1-cm. optical paths were used for all measurements. The apparatus for electrophoresis, using agar as a stabilizing medium, has already been described (7). PROCEDURE
Normal sera for these experiments were contributed by six men and six women ranging between the ages of 24 and 47 years. Electrophoreses were conducted in 1% agar gels of diethylbarbiturate buffer (pH 8.6, ionic strength 0.1) by applying a constant 2.5 volt per cm. potential gradient for 24 hours. The separate fractions were collected by trichloroacetic acid precipitation according t o techniques already described (7). Diethylbarbituric acid was removed by washing the precipitates twice with 5% trichloroacetic acid; then each fraction was dissolved in a 10-ml. portion of carbonate-free 0.lN sodium hydroxide. Except for the albumin-1 fraction, which required fivefold dilution, no further preparation was needed for ultraviolet spectrophotometry. Absorbance due to trichloroacetic acid associated with the proteins was found to be negligible in alkaline solution. The solutions were scanned between 225 and 340 mw, using a 0.1N sodium hydroxide blank; then aliquots were taken for Kjeldahl nitrogen determinations by the micromethod. Replicate determinations showed that the precision of the nitrogen determination allowed errors not to exceed *5.6oJ, within 99% confidence limits. INTERFERING SUBSTANCES
When proteins are separated from
agar by the method recommended, trichloroacetic acid remains associated with the precipitates. To determine the quantitative relationship between the proteins and acid, 2 X 5 X 10 mm. agar blocks were impregnated with protein by immersion in normal sera for 24 hours. This protein was separated from the blocks and the bound acid estimated by obtaining the titration curves of the precipitates us. 0.0100N sodium hydroxide. Following titrations, the protein content of each titration mixture was determined directly. I n this way it was established that serum proteins bind 6.27 X 0.07 X 10-3 (std. dev., six sera) mole of trichloroacetic acid per gram of protein nitrogen. The molar absorptivities of trichloroacetate in 0.1N sodium hydroxide a t the applicable wave lengths were determined and the extinctions attributable to protein bound trichloroacetic acid were calculated (Table I). These data indicate that trichloroacetic acid precipitation should affect the absorption spectra of serum proteins negligibly. This was confirmed by comparing the spectral curves in 0.1N sodium hydroxide of aliquots of serum separated from agar with identical aliquots of untreated serum, No significant differences n-ere found between the spectra of the untreated and the precipitated proteins. The possibility remained that the process for freeing the proteins from agar might cause loss, by hydrolysis, of some light-absorbing components such as tyrosine. To test this, the supernatant liquids obtained by melting the agar, adding trichloroacetic acid, and packing the proteins by centrifugation were scanned using blanks made to the same concentration of trichloroacetic acid and agar. These preparations exhibited no absorbance in the regions of the tyrosine or tryptophan maxima and no significant absorbance over the
*
Table 1.
Effect of Bound Trichloroacetic Acid Upon Ultraviolet Absorption Spectrum of Serum Protein R a v e Length, M p 225 230 235 240 245 250 255 260 Molar absorptivity of trichloroacetic acid 1944 1013 5 0 6 253 106 7 4 3 8 1 9 Absorbance per gram of protein nitrogen attributable to bound trichloroacetic acid 1.22 0 . 6 4 0 . 3 2 0 . 1 6 0 07 0 . 0 5 0 02 0 01
VOL. 32, NO. 2, FEBRUARY 1960
199
entire spectral interval between 225 and 340 mp. Agar displays a weak generalized ultraviolet absorption, but the amounts remaining associated with a protein precipitate after the ivashing procedure are below the limit of spectrophotometric detection. This has been confirmed indirectly: The nitrogen factors of proteins precipitated from agar were found not to differ from those of proteins precipitated in the absence of agar. This would not be true if agar, a nonnitrogenous polysaccharide, associated appreciably to proteins. Diethylbarbituric acid has a n absorption maximum a t 255 mp that could interfere n i t h the determination of protein spectra if it remained associated n ith the precipitated proteins. Diethylbarbiturate, however, is not bound to serum proteins in the presence of trichloroacetic acid. This fact n a s demonstrated by separating the protein fractions from electrophorogranis prepared in the prescribed manner, pooling the fractions, and determining their barbital content by Broughton's method ( S ) , I\ hich employs ultraviolet spectrophotometry and is capable of detecting 0.5 y of diethylbarbituric acid. Three electrophorograms, analyzed in this n a y , contained no barbital.
VI
f
0)
5
-I
m
; VI
.-5 L
9 c
0
.-2a
c
P
0-
RESULTS A N D DISCUSSION
Serum proteins were separated into 11 fractions by electrophoresis. These fractions all dissolved readily in 0.LV sodium hydroxide. The ultraviolet spectra of the various fractions were found to differ from each other, although all conform to the general curve which has been described. Table I1 shows the results, a t intervals of 5 mp, expressed I 1 as the absorptivity, E = -log Io X bc I where -log - represents absorbance 10 of the solution, b is the length of the optical path in centimeters, and c is the concentration of KJeldahl nitrogen in grams per liter of solution. The means and standard deviations of the absorptivities for all fractions a t a n y given wave length (Table 11) indicate that little reliance can be placed upon estimations of mixed serum proteins by simple ultraviolet absorbance measurements. Even a t 245 mp, the n a v e length a t 17 hich the absorbances of the different fractions are most alike, the standard deviation for individual fractions is 9.6% of the mean. Comparison of the absorptivities a t 245 nip of corresponding electrophoretic fractions from different individuals (Table 111) makes it evident that even specific fractions cannot reliably be estimated a t this wave length. TF'ave lengths other than 245 mp offer even less promise for such determinations. Raddell (8) has suggested estimating 200
ANALYTICAL CHEMISTRY
E
2m
v) q_
0
m
U
6
-?! SI
2
-a0 .-> 0 3 -m c
c L
1)
I-
0
Individual Variations in Absorptivity at 245 Mp of Proteins Separated from Serum by Electrophoresis
Table 111.
Serum
Rho
1 2 3 4 5 6
33.2 49.8 11.9 35.2 12.5 73.2 14.0 33.8 31.1 37.6
m
L9 10 11 12
...
RIean Std. dev.
33.4 33.24 17.76
Alpha*-1 Alpha*-2
Albumin-1 A41buniin-2 alphal ... 44.4 37.1 ... 39.3 37.8 27.5
28.8
...
...
32.8
46.8 42,9
...
33.1 33.0 31.8 21.7 36.3 31.4 31.13 4.35
...
37.0 43.2 23.5 42.2 54.6 40.14 9.06
42.4 t
.
38.0 38.5 43.0 40.1 33.5 35.3 47.3 38.0 36.9
34.6 37.7 50.0 34.8 32.2 33.5 56.3 42.1 33.3 28.8 28.8 29.4 36.78 8.63
.
56.1 35.2 45.8 40.4 42.3 28.5 76.3 33.3 43.20 13.12
-
Beta
Gammal
Gamma?
41.6 36.9 40.9 44.0 29.0 28.9 33.9
64.7 32.0
42.1 41.8
... .
...
37.9 42.8 37.6 36.0 37.20 5.10
...
I
.
27.5 30.4 31.6 42.5 19.4 61.1 38.3 15.27
30.2 34.8 64.6 29.6 36.7 62.3 19.4
43.8 39.1 57.9 39.0 35.7 35.2 34.9 42.3 36.7 52.2 30.0 53.0 41.89 8.63
.
61.6 30.6 35.1 22.0 42.4 33.1 45,4 35.1 47.5 39.70 10.36
.
35.3
...
39.5 45.0 39.55 4.13
I
Gamma3 Gamma4
...
30.2 53.5 43.9 46.3 41.03 14.46
Table IV. Tyrosine and Tryptophan in Serum Proteins RIeans with standard deviations for fractions separated from 12 sera Albumin- hlbuminiilpha2- AlphanTotal Rho 1 2 Alpha1 1 2 Beta Gammal Gamma2 Gamma3 Gammaa Protein
Tyrosine, nimoles per gram of Kjeldahl nitrogen
2.58 2.68 2.74 2.30 2.56 2.65 2.62 2.34 2.31 3.18 2.81 2.69 f 1 . 2 3 1 0 . 8 2 1 0 . 8 8 1 0 . 6 9 1 0 . 3 7 h 0 . 3 2 1 0 . 4 2 9 ~ 0 . 5 0 rt0.52 1 0 . 8 0 1 1 . 1 9 1 0 . 4 1 0.79 1.43 1.48 1.64 1.42 1.44 1.28 1.16 Tryptophan,mmolesper 1.11 1.53 1.41 0.72 gram of Kjeldahl nitro- 1 0 . 6 9 1 0 . 2 3 f 0 . 5 3 1 0 . 3 9 1 0 . 9 7 1 0 . 1 6 f 0 . 1 8 1 0 . 8 5 1 0 . 4 9 1 0 . 4 2 1 0 . 5 6 1 0 . 4 3
gen Rlean molar ratios of tyrosine t o tryptophan
2.44 3.49 2.10 2.00 2.00 2.29 1.89 1.65 1.75 1.96 2.17 3.32 1 0 . 4 9 f 0 . 5 0 h 0 . 2 3 1 0 . 2 3 h 0 . 2 9 1 0 . 3 1 AO.18 h 0 . 4 6 f 0 . 4 1 1 0 . 2 2 1 0 . 2 3 h 0 . 4 4
proteins by multiplying a constant times the difference betn een their absorptions a t 215 and 225 mp. This has been endorsed hy Bendixin ( 2 ) . When this method n as applied to electrophoretically separated fractions, correspondence of the optical measurement to nitrogen content n a s found to be even worse than for single wave length nieasurements. The \Tide differences among the ultraviolet spectra of the various protein fractions (Table 11),as well as betneen corresponding fractions from different serum samples (Table III), indicate that the fractions arc’ mixtures rather than pure proteins. This view is supported by the range of variation in their tyrosine and tryptophan contents as determined by the method of Holiday (6) (Table IT). The present authors’ determinations of the molar absorptivities of tyrosine (€280 m,, = 1570; €294 1 = 2440) and of tryptophan (eso m,, = 5210; mii = 2440) differ slightly from those accepted by Holiday so that the coefficients for the Goodn in and Morton formulas (4) differ from those used by Holiday. The formulas, using the authors’ coefficients are AI,,, A1tn,,
= =
0.588 €29, 0 275
4 mp
€z.O m p
- 0.275 €280 - 0 176
mfi
(1)
€294 4 mp
(2)
where e refers to absorptivities a t the indicated wave lengths and M,,, and Mt,,p are the millimolar concentrations of tyrosine and of tryptophan in the sample solutions. The distribution of tyrosine and tryptophan among the various fractions. and for total serum
Table V.
Sex Differences in Tyrosine and Tryptophan Content of Albumin-1 Serum Protein Fraction
(Quantities of amino acids expressed as mmoles per gram of protein nitrogen) Tryptophan Tyrosine LIales ( y) Females ( 2 ) Males ( y ) Females
Mean Std. dev. hIean, males and females combined Std. dev., males and females combined
2 3 3 2 2 3 3 1 0
2 2 1 1 2 3 2
04 07 94 62 16 46 21 1 0 64
01 76 47 89 86 97 16 72
0 0 0 0 0 1 0 1 0
(E)
0 76 1 76 0 95 0 88 0 92 1 50 1.13 f0.40
67 5G 55 38 53 02 62 22
2 69
0 Si
1 0 82
1 0 41 t = 3.08
Critical value of t at 270Fprobabilitylevel (10 D.F.) proteins, is shown in Table Is’. Yariations between the different fractions are too large to permit accurate estimations of serum proteins by their aromatic amino acid content. Henitt (6) has reported the separation of albumin into ta o fractions by salting out n ith amnionium sulfate. One of these fractions is low in tryptophan i Z $ E h a n = 25) while the second fractibh h k a higher tryptophan content. The tyrosine and tryptophan ratio of albumin-1 (Table IV) is higher than that of the other proteins, indicating a relative deficiency of tryptophan, but does not approach the 25 to 1 ratio. This indicates that the tFvo albumin fractions of Hewitt are not separated by electrophoresis.
(
=
2.764
Attempts to do so by extending the length of the electrophoretic run failed to &on any indication of fractionation into albumins differentiable by their tyrosine and tryptophan content. When the portion of agar containing the albumin-1 fraction n a s cut in half so that the more rapidly moving portion of the albumin could be analyzed separately from the slon er portion, the t n o halves did not differ in their tyrosine-tryptophan ratios. The composition of the albumin-1 fraction may vary n i t h respect to sex (Table Y). This observation is based on analyses of only 12 sera from 6 persons of either sex; even so, Student’s t test gives confidence above the 98% level that the albumin-1 fraction from VOL. 32, NO. 2, FEBRUARY 1960
201
women is comparatively rich in tyrosine and tryptophan. This difference may be related to the metabolism of the estrogenic hormones; more work will be required before this can be elucidated. KO other protein fraction displays significant sex variation m-ith respect to aromatic amino acid content. LITERATURE CITED
(1) Beaven, G. H., Holiday, E. R.,
Advances i n Protein Chem. 7, 319-82 (1952). (2) Bendixin, G., Nord. M e d . 5 8 , 1488 (1957). (3) Broughton, P. M. G., Biochem. J. 6 3 , 207-13 (1956). (4) Goodwin, T. W., Morton, R. A., Ibid., 40,628-32 (1946). ( 5 ) Hewitt, L. F., Ibid., 30, 2229-36 (1936). (6) Holiday, E. R., Ibid., 30, 1795-1803 (1936). ( 7 ) Strickland, R. D., Mack, P. -4.,
Gurule, F. T., Fodleski, T. R., Salome, O., Childs, W. -4.,-4iv.4~.CHEM.31, 1410-13 (1959). (8) Waddell, W, J., J . Lab. Clzn. M e d . 48, 311-14 (1956).
RECEIVEDfor review July 23, 1959. Accepted November 2, 1959. Investigation supported in part by research grant (H2100) from National Heart Institute, National Institutes of Health, Public Health Service, Department of Health, Education, and Kelfare.
Potassium Phosphate as a Reagent for the Gravimetric Esti mati o n of Lithium C. C. PATEL
and
K.
N. VISHWESHWARAIAH
Department of lnorganic and Physical Chemisfry, Indian Institute o f Science, Bangalore 12, India
b Previous aftempts for the quantitative estimation of lithium as orthophosphate, employing an alkali metal phosphate, have not been successful. A method is described for the estimation of lithium as trilithium phosphate from 60% ethyl alcohol solution a t 65' to 70' C., employing potassium phosphate reagent at p H 9.5. The method is applicable in the presence of varying amounts of sodium and/or potassium cations and chloride, sulfate, nitrate, and phosphate anions.
L
as trilithium phosphate was determined previously by Mayer ( 5 ) ,who used an aqueous alkaline medium and a reagent consisting of a mixture of sodium phosphate and sodium hydroxide; however, Rammelsberg (6) and Fresenius (3) found that coprecipitation of sodium phosphate invariably occurred with the precipitates of trilithium phosphate. Furthermore, lithium phosphate was found to be appreciably soluble in the aqueous ammoniacal solution used for washing the precipitates. Recently, Caley and Simmons (2) attempted to modify Mayer's method by employing a n alcoholic medium to precipitate lithium as phosphate at about 65" C., but the method did not succeed. However, choline (a quaternary ammonium base) phosphate was successfully used by Caley and Simmons for the precipitation of lithium as orthophosphate from 50% isopropyl alcohol, but these authors pointed out that it was difficult to get a pure quaternary ammonium base suitable for the preparation of the phosphate reagent. I n place of choline phosphate, Vishweshwaraiah and Pate1 (7) employed ITHIUM
202
ANALYTICAL CHEMISTRY
the phosphate reagent of 8-diethylaminoethyl alcohol and obtained good accuracy for the gravimetric estimation of lithium. The purity of the precipitate was very high, even when it was precipitated from 60% ethyl alcohol in the presence of tripotassium phosphate. This suggested the possibility of employing the common reagent potassium phosphate for the quantitative precipitation of lithium in place of other expensive organic phosphates. Potassium phosphate is particularly suitable for the estimation of lithium, because its solubility in 60% ethyl alcohol is more than that of sodium phosphate. d study was undertaken to find out the limitations of using potassium phosphate for the gravimetric estimation of lithium. Initially, potassium phosphate reagent from p H 8 to 10 was tried, but the reagent a t p H 9.5 gave the best results for the precipitation of trilithium phosphate and was therefore used for subsequent work. EXPERIMENTAL
Reagents. LITHIUM CHLORIDE. A neutral solution of lithium chloride was prepared from lithium carbonate, purified by t h e method of Caley and Elving (1). ORTHOPHOSPHORIC ACID. Guaranteed Dure Merck Droduct of specific gravitj- 1.75. ETHYL ALCOHOL. Absolute ethvl alcohol was further purified by the method of Lund and Bjerrum (4). POTASSIUM HYDROXIDE, Analytical reagent quality pellets were employed. TRILITHIUM PHOSPHATE.Prepared by reacting lithium chloride solution with potassium phosphate as described in the procedure. The precipitate was washed free from potassium ions by 60% alcohol and dried a t 110" C. for about 24 hours.
WASHLIQUOR. Clear saturated solution, obtained by shaking 60% ethyl alcohol (by volume) with pure trilithium phosphate for about 12 hours a t room temperature, was employed for mashing the precipitates of trilithium phosphate. PHOSPH.4TE REAGENT. One hundred milliliters of 8% aqueous solution of potassium hydroxide were mixed 11-ith the required amount of orthophosphoric acid till the p H of the resulting solution was 9.5. PROCEDURE
The procedure employed is practically the same as the one employed in the determination of lithium as phosphate, using the phosphate reagent prepared from 8-diethylaminoethyl alcohol (7). Lithium chloride solut,ion (10.0 nil.) containing 5 to 50 mg. of lithium was made to 14 ml. nith distilled vater and mixed with 6 nil. of the potassium phosphate reagent in a borosilicate glass beaker of 250-ml. capacity. The beaker n-as covered with B borosilicate glass dish containing cold water, xi-hich was frequently changed in order to minimize the losses of vapors during heating. The mixture was placed on a water bath maintained between 65" and 70" C. One hour after the first appearance of the precipitate, 30 ml. of absolute ethyl alcohol were added to t h e beaker, xhile stirring, to make the concentration of the alcohol 60%. The heating ivas continued for another 2 hours after the granulated precipitate was obtained. The hot solution was rapidly filtered through a medium porosity porcelain sintered crucible that was previously ignited, cooled, and weighed. The precipitate n a s washed successively with small quantities of wash liquor until about 50 ml. of it were used, after which air was sucked through the precipitate to remove most of the n-ash liquor.
