Determination of Protein-Bound Carbohydrates by the Anthrone Reaction Effect of Tryptophan ELIZABETH F. TULLER and NILS R. KEIDING‘ George
F. Baker Clinic Research Laboratory, N e w England Deaconess Hospital,
Mass.
reported as milligram per cent in the solution prior to addition of sulfuric acid. All Cohn fractions and purified protein PROTEINSOLUTIONS. solutions were 200 or 400 mg. % in 0.85% saline. With one or two of the fractions, such as Fraction 111, it was necessary to add a small amount of 0.5% sodium hydroxide and to heat gently to effect complete solution of the protein. ~\‘INZLER ~ ~ U C O P R O T E I SPRECIPITATE (11). The procedure used for separating the Winzler precipitate was essentially t h a t of the original workers. For this work, the resultant precipitate must be washed with ethyl alcohol to remove traces of the phosphotungstic acid which may interfere with the anthrone reagent. TOTAL SERUXPROTEIN PRECIPITATE. An alcohol precipitate of serum protein was made by adding 0.5 ml. of a 1 to 10 dilution of serum to 10 ml. of alcohol. The suspension was allowed to stand 10 to 15 minutes before it was centrifuged. Method. The determination was carried out by adding 0.5 ml. of anthrone solution to 2 ml. of a carbohydrate-containing solution in a 20 X 150 mm. test tube. Five milliliters of concentrated sulfuric acid were carefully layered into this tube, which was first gently and then rapidly SR irled to hydrolyze the ethyl acetate and completely mix its contents. For maximum stable color development, all solutions mere allowed to stand for 0.5 hour and not more than 1.5 hours before readings were taken in a Beckman D U spectrophotometer. For a carbohydrate standard curve only the wave length a t 630 mp was used. For all other work wave lengths were used as indicated in the tables and the figures. I n the determination of the nonhexosamine carbohvdrate in protein precipitates by this method the precipitates ”were dissolved in 10 ml. of 4.4% sodium carbonate solution and 2 ml. of the resultant solution were used. If hydrolysis of the proteins mas carried out, 2 mi. of the protein solution were refluxed with 2 ml. of %IT hydrochloric acid for 4 hours. The resultant hydrolyzates were then diluted to 10 ml. and 2 ml. of the diluted solutions were used for the carbohydrate determination.
This investigation was undertaken to determine the cause of and the possible effect of the appearance of a 530-mp absorption peak in the quantitative determination of carbohydrate in protein-containing materials by the anthrone method. The principal cause of this absorption peak was shown to be the formation of a product (TSA complex) from the tryptophan and sugar in the presence of excess anthrone and sulfuric acid. In order to eliminate the 5 to 15% error in the estimation of carbohydrates caused by the presence of tryptophan in protein solutions, a nomogram prepared by plotting the ratio of the absorbancies of the 530-mp peak to the 630-mp peak of the spectrum of known mixtures against the absorbancy of the 630-mp peak may be used.
T
HE anthrone reaction for the qualitative and quantitative analysis of carbohydrates has been used by many workers since the original studies of Dreywood (1). It has been observed that in the presence of proteins, in addition to the 630-mp peak caused by the carbohydrate-anthrone reaction, there is another maximum a t 530 mp ( 2 ) 7 , 9, I O ) . I n the course of a study of the serum protein fractions, done prior to the publication of Shetlar’s work ( I O ) , giving definite evidence that the absorption peak a t 530 mp resulted from the presence of tryptophan, it was apparent that whatever caused the 530-mp peak also might be interfering with the accuracy of the analysis for saccharides. It was believed that tryptophan, a lipide material, or both might contribute to this absorption peak. The possibility of an effect from lipide material was strengthened by the work of Graff et al. (S), as i t was apparent from this that cholesterol would react with the anthrone reagent. This study was initiated in an attempt to clarify the behavior of the 530-mp absorption peak and to learn the possible effect of the reaction producing this absorption on the determination of carbohydrates in proteins or protein-containing materials by the anthrone reaction. The results confirm Shetlar’s work ( I O ) on the cause of the 630-mp maximum and, in addition, show definitely that it is possible, in the absence of large quantities of lipides, to predict the behavior of protein-containing materials i n this determination. A method was also devised to correct for the presence of tryptophan, which may cause low values in the determination of carbohydrates.
RESULTS
Reactions of Protein with Anthrone Reagent. Protein reactions with the anthrone reagent appear to fall, for the most part, into general types as illustrated by the typical curves given in Figure 1. One type shows a weak maximum absorption a t 530 mp and little or no absorption of 630 mp (Figure 1, A ) . Cohn Fractions I, 111, and V belong to this group. A second type shows absorption a t both wave lengths and includes Cohn Fractions 11, IV-1, and 117-4, egg albumin, and an alcohol precipitate of the serum proteins (Figure 1, B ) . A few proteins shorn. only an absorption band a t 630 mp, while others show no absorption a t either 530 or 630 mp. An example of the former is Schmid’s (8) acid glycoprotein (Figure 1, C) and of the latter, electrophoretically pure serum albumin. I n examining the absorption spectra of a number of purified proteins, one in particular was found to give a completely different picture from any described above. The &-lipoprotein (Figure 1, E ) shows a slight plateau from 460 to 490 mp and no evidence of either a 530- or 630-mp absorption peak. This protein is reported to be composed of 2501, protein, 45y0 cholesterol, and 30% phospholipide (4). An examination of the spectral curves for cholesterol and a phospholipide, lecithin, indicated t h a t the plateau in the 460- to 490-mp region of the lipoprotein spectral curve may be attributed to cholesterol. These lipide materials of the protein may be extracted by an alcohol precipitation of the protein and the new curve becomes very similar to t h a t of the blank except for a slight absorption a t 630 mp, indicating the presence of a small amount of carbohydrate. Alpha-lipoproteins apparently react in the same manner as
EXPERIMENTAL
Reagents and Materials. ANTHRONESOLUTION.A slight modification of Loewus’ ( 5 ) procedure was used for the preparation and use of this reagent. A 1% solution of anthrone (Microne reagent, National Biochemical Co.) was made up in reagent grade ethyl acetate. The solution must be made up a t least 4 hours before use or, preferably, allowed to stand overnight. If kept in the refrigerator i t will remain stable for a week to 10 days. A change in blank reading is an indication of a change in the reagent. CARBOHYDRATE SOLGTIOXS.Unless otherwise indicated, all carbohydrate solutions used in this work were equimolar mixtures of galactose and mannose. Concentrations are given as milligram per cent of total sugar per 100 ml. of solution before the addition of sulfuric acid. TRYPTOPHAN SOLUTIOSS. Aqueous 1-tryptophan solutions are 1 Present
Boston,
address, S t e m Memorial Hospital, Gentofte, Denmark.
875
ANALYTICAL CHEMISTRY
876
is tryptophan completely removed under the conditions used. Thus, while trypto+ phan is more readily destroyed by acidic C -ACID GLYCOPROTEIN hydrolysis when in the presence of carboD -0- FRACTION I p - l hydrates ( 6 ) , the actual amount of the E --Cr B -LIPOPROTEIN destruction is dependent on concentraF *REAGENT BLANK tions and may not be complete under the conditions necessary to hydrolyze the protein entirely. Other Substances and Anthrone Reaction. A number of other substances were examined to see if some other possible contaminants or constituents of either the Cohn fractions or serum would produce an absorption band at 530 mp in the anthrone determination of carbohydrates. Segative results were found with desoxycorticosterone acetate, glucosamine, cupric ion, ferrous and ferric ions, and pentoses, although some of these showed bands a t other points. Under conditions of the determination reported WAVE LENGTH, rnp here, ascorbic acid in concentrations up to Figure 1. Spectral Curves of Proteins Following Reaction with the Anthrone 5 mg. yo in the solution to be analyzed Reagent gave no more absorption than a reagent blank. The effects of dl-tryptophan, . . phenylalanine, and tyrosine on the carbohydrate-anthrone deterthe pl-lipoqrotein, since Fraction IV-1, which contains approximination were tried. DI-tryptophan reacted identically as I-trypmately one third orl-lipoprotein, showed some of the spectral tophan, and phenylalanine had no effect. Preliminary work with properties of both the pl-lipoprotein and the globulins. As may tyrosine does indicate that it may resemble tryptophan in its be seen in Figure 1, D, Fraction IV-1 shows a slight 530-mp peak but also remains rather highly absorbant in the 460- to 490-m,~ effects, although to a far lesser extent-Le., slight plateaus are produced in the 530-mp region with a diminution of the 630-mp peak. region, in contrast to purified 7-globulin or the total serum proThis effect should be investigated in greater detail. Determinatein. An alcoholic precipitation of Fraction IV-1 followed by tions done with sugars other than galactose and mannose indicate the anthrone reaction apparently resulted in a removal of lipide that in the presence of tryptophan similar results would be obmaterial, since the resultant curve showed no plateaus, no 530-mp tained, although the effect o n both the 530- and the 630-mp peak, and essentially the same absorption in the 630-mp region. peaks is different in intensity. Reaction of Tryptophan with Anthrone Reagent in Presence of Carbohydrates. The addition of even minute amounts of carboDISCUSSION hydrate to solutions containing tryptophan resulted in a marked The interrelationship of all the spectral curves in Figure 2 increase in absorption a t the 530-mp wave length, in contrast to seen1 to indicate that an equilibrium reaction has taken place. the slight absorbancy noted with tryptophan solutions treated The colored substance (A maximum 530 mp) resulting from the with the anthrone reagent. When large amounts of tryptophan were added to constant amounts of sugar, the absorbancv a t 530 mu seemed to approach a maximum value, as has since A .-.e4.1 mg.% GALACTOSE-MANNOSE SOLUTION been reported by Shetlar (IO). When B -!+ 20 mg.% TRYPTOPHAN I N A this effect was studied quantitatively, the C -e-I O mg.7. TRYPTOPHAN I N A resultant curves show a regular decrease D 4 - 2 5 mg.Y. TRYPTOPHAN I N A in the 530-mp peak with an increase in E -*-20 mp.% TRYPTOPHAN the 630-mp peak, and all curves cross a t 05 approximately 580 mp. Representative curves of 4.1 mg. % galactose-mannose solutions are given in Figure 2. 0.4Solutions containing various amounts z m a of tryptophan or of electrophoretically 0: u) 0 pure 7-globulin and a constant amount 0.3of sugar were hydrolyzed for 4 hours in order to determine qualitatively the completeness of the destruction of tryptophan 0.2in the presence of carbohydrate under these conditions. Since the height of the 530-mp peak is dependent primarily on the molar ratio of tryptophan to sugar, the absorbancies after hydrolysis should 7 be less than before if destruction had I 500 520 540 560 580 600 620 640 660 680 taken place; such was the case (Table I). WAVE LENGTH, mp As may be seen from the absorbancies Figure 2. Effect of Tryptophan on the Spectral Curves of Galactose-Mannose in the -4nthrone Determination given, only in very low concentration A-n-FRACTION I B TOTAL SERUM PROTEIN
z
V O L U M E 26, NO. 5, M A Y 1 9 5 4 Table I. Hydrolysis of Tryptophan and y-Globulin in Presence of a Constant .4mount of Sugar (5 IIg (3 galactose-mannose) Absorbancies Absorbancies after Hydrolysis, Solution Conon.. heforr Hydrolysis“, 530 M p hfg. 70 530 I l p 0 475 0 183 2 0 . 0 tryptophan 0.375 0 176 1 0 . 0 tryptophan 0.310 0 116 5 . 0 tryptophan 0,290 0 100 2 . 5 tryptophan 200.0 r-Globulinb (5.6 0 250 0 147 % tryptophan) ‘ 160.0 yglobulm (4.2 0 136 0.228 % tryptophan) 100.0 7-globulin (2.8 0 114 0 210 % tryptophan) 2 5 . 0 -,-globulin (0.7 0 .180 0 089 70 tryptophan) 5 0 y-globulin (0.14 ( I . 170 0 074 70 tryptophan) 0 073 0 075 Pure galactose-mannose ‘1 Since hydrolyzed solutions were diluted, absorbancies for t h e corre+pending iinhydrolszetl solutions were read froin a standard curve. b Gamma-globulin contains a~?proriniately2 . 8 grams of t,ryptophan per 100 grams of protein. Approximate tryptophan concentrations for the yglobillin solutions were calciilated.
877 yses and hence, destroy one of the advantages of the anthrone method. A second way of handling the problem of analysis of protein-containing material is to use a monogram which will correct for the tryptophan effect regardless of a considerable variance in dilution of the original material and regardless of the variance in the concentrations of tryptophan and sugar. This method was Cied and Figure 3 shows a monogram in which the ratio of the height of the 530-nip peak to that of the 630-mp peak is plotted against the 630-mp reading. These coordinates were adopted in preference to plotting the ratio against the 530mp reading, because the 630-nip reading gave more reproducible results. Each line then represents :I different concentration of sugar and each point on a line represents a different molar ratio of tryptophan to sugar. The publication by Shetlar of his investigation of the ant,hrone reaction (IO) called the authors’ attention to the direct use of the 530-mp absorption peak. Their own experience thus far with t,his method confirms their general experience with the use of the 530-mp absorption peak-i.e., results are less reproducible. Evidently, the peak a t 530 mp of the absorption curve of the TSA complex is more sensitive and, hence, harder to reproduce, a t least under the conditions of these studies.
interaction of tryptophan, sugar. sulfuric acid, and anthrone is here designated for convenience ai; t,hc TS.4 complex. I n the iresence of an excess of sulfuric ilcid and ant,hrone, the concen4PPLICATIONS OF NOMOGRAM METHOD trations of this complex are depend(~ntupon the relative concenThe carbohydrate content of the Winzler mucoprotein fraction tration of tryptophan to sugitr. :\pparently, the complex has and the “total protein-bound nonhexosamine carbohydrate” of maximal absorption at about the same wave length as do pure serumwerestudied by meansof the nomogrammethodoutlined. As tryptophan and anthrone (520 nip), but the complex shows far may be noted in Table 11,the carbohydrate content of the Winzler greater absorbancy. Such an equilibrium reaction would explain mucoprotein in the first three samples may be read usually thc difference noted by S h d a r (IO) between the unexpectedly with equal accuracy from an ordinary standard curve or from high absorption peak a t 530 nip for a mixture of tryptophan, the nomogram. The absence of the tryptophan effect (despite carbohydrate, and anthrone and the low absorption in the theoretthe known tryptophan content) was confirmed by hydrolysis ical curve calculation from absorbance measurements of the which produced no change in the observed carbohydrate values. individual components. This ecjuilibrium would also account for On the other hand, in a case in which a high gamma-globulin conthe various absorption curves of the proteins already observed tent in the serum protein was indicated by paper electrophoresis, and would allow for the prediction of the expected reaction of any as in the fourth example, the Winzler precipitate showed a small ne^ mixture which contained tryptophan and carbohydrate. 530-mp peak. A standard curve reading, therefore, gave values That this prediction may be made when lipide materials are not only 92% of what it should have been according to the monogram present in high concentrations is shown by an examination of and according to results after h!Tdrolysis. I n the samples of the curves in Figure 1. For example. an alcohol precipitate of total serum protein of the total protein-bound nonhexosamine the serum proteins or of Fraction I containing both tryptophan and carbohydrate in moderate quantities gives well-defined absorption peaks :it mg,% GALACTOSE - M A N N O S E both 530 and 630 mp. On the other hand, proteins such as the mucoproteins which contain only small amounts of tryptophan but extremely h r g e amounts of carbohydrat
