Analyst, January, 1973, Vol. 98, @. 25-33 25 Effect of Isoelectric Focusing on the Amino-acid Composition of Proteins BY S. JACOBS (National Institute for Medical Research, Ridgeway, Mill Hill, London, N . W. 7) The normal isoelectric focusing method has been used to separate various proteins into fractions. The fractions and the parent proteins have been hydrolysed and the amino-acid composition of each has been determined by ion-exchange chromatography. Evidence was obtained that shows that the parent protein is modified by the normal isoelectric focusing procedure; the sulphur-containing amino-acids cystine and methionine were shown to be present partly in the form of cysteic acid and methionine sulphoxides, in a greater proportion than with hydrolysates of proteins isolated by other separation procedures.Prolonged treatment of the protein by the isoelcctric focusing procedure adversely affects other amino-acid residues in the molccule also. An improved method has been devised so as to protect the protein isolated by the isoelectric focusing procedure and to prevent the oxidation or modification of the protein, which would otherwise occur during the normal isolation procedure. THE theoretical principles of isoelectric focusing were demonstrated by Svenssonl in 1961 , and Vesterberg and Svensson2 developed a series of synthetic ampholytes suitable for produc- ing a natural equilibrated pH gradient. A sucrose gradient was used to prevent convective disturbances .3 The technique of isoelectric focusing has been used by several workers to isolate different proteins that displayed heter~geneity~--~ when examined by this method.The ion-exchange resin chromatography of the hydrolysates of proteins, isolated by the normal isoelectric focusing procedure, has been performed in this laboratory. In each instance, the amounts of cysteic acid and methionine sulphoxides present in the hydrolysates of proteins isolated by isoelectric focusing exceeded those in proteins isolated by other separation procedures. The amino-acid compositions of component fractions of the proteins thyrotrophin, or-glycoprotein and bovine ribonuclease have been determined, and the results showed that the protein was modified during the normal isoelectric focusing procedure. The latter procedure has therefore been changed in order to prevent such alterations in the amino-acid composition of the isolated protein. EXPERIMENTAL MATERIALS- The ion-exchange resins that were used to examine the hydrolysates of proteins separated by the isoelectric focusing procedures were supplied by Permutit Company Ltd.and Beckman Instruments Ltd. The bovine ribonuclease A, Grade V (chromatographically pure), was supplied by Miles Seravac Ltd., Maidenhead, Berkshire. The ampholine ampholytes used for producing the natural pH gradients in the sucrose gradient columns for isoelectric focusing were supplied by LKB Produkter AB. REAGENTS- were prepared with conductivity water as previously described.10 The properties of these resins were given in a previous paper.9 All reagents were of analytical-reagent grade unless otherwise stated, and all solutions Indanetrione hydrate, normal laboratory grade. Indanetrione hydrate stock solution.Indanetrione hydrate reagent. Methyl Cellosohe (2-methoxyethanol) , technical grade. Sodium acetate trihydrate. Sodium acetate bufler solution, 4.0 M, +H 5.5. The above six reagents were prepared as described previou~ly.~ @ SAC and the author.26 JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THE [Anahst, vol. 98 Sodium citrate buffer solution, 0.2 M, p H 3.25-This was produced in bulk amounts of 20 litres for use with the Beckman amino-acid analyser. Each batch of 20 litres of buffer solution contained 160g of sodium hydroxide, 420g of citric acid, about 214ml of concen- trated hydrochloric acid, 1.0 g of Brij 35, 200 ml of 2,2'-thiodiethanol (thiodiglycol) and 2 ml of octanoic acid, The octanoic acid was of laboratory-reagent grade. Sodium citrate buffer solution, 0.2 M, @H 4.25-This buffer solution was produced in bulk amounts of 10 litres for use with the Beckman amino-acid analyser. Each batch of 10 litres of buffer solution contained 80 g of sodium hydroxide, 210 g of citric acid, about 47 ml of concentrated hydrochloric acid, 100 ml of 2,2'-thiodiethanol, 1.0 g of Brij 35 and 1.0 ml of octanoic acid.Sodium citrate b u f e r solzttion, 0.35 M, j5H 5.28-Each batch of 20 litres of this buffer solution contained 280 g of sodium hydroxide, 491 g of citric acid, approximately 135 ml of concentrated hydrochloric acid, 2 g of Brij 35 and 2 ml of octanoic acid, but contained no 2,2'-thiodiethanol because no sulphur-containing amino-acids were determined during the elution of amino-acids on the column used to elute basic amino-acids.11 APPARATUS- The isoelectric focusing was performed on the LKB standard equipment with the 8100 elect rof ocusing column. Some comparative isoelectric focusing experiments were performed on the preparative density gradient electrophoresis apparatus supplied by Instrumentation Specialities Co.Inc. (ISCO), Lincoln, Nebraska, USA. The amino-acid analyses of the protein hydrolysates were performed on a Beckman Amino-Acid Analyser, Model 120C, by using the two-column method previously described12 with ion-exchange resins of improved q ~ a l i t y . ~ PROCEDURE- The isoelectric focusing was performed on 10 to 12-mg amounts of the ribonucIease A by using the standard LKB or the ISCO apparatus.The LKB 8100 electrofocusing column was maintained at 5 "C. The sucrose gradient was adjusted so as to have a concentration that varied from 50 to 5 per cent. of sucrose, and the solution surrounding the cathode con- tained 50 per cent. of sucrose and 1 per cent. of tetraethylenemethylenediamine. The ampholine ampholytes had a pH in the range 7 to 10. The total volume of the solution containing the sucrose gradient was 110 ml, and the concentration of the ampholine in this gradient was 1 per cent. A 10-ml volume of aqueous solution containing the antioxidant was introduced carefully so as to form a layer above the sucrose gradient. The 10ml of 10 per cent. solution of ascorbic acid contained no ampholine, whereas the 10ml of either 10 or 5 per cent.2,2'-thiodiethanol as antioxidant contained 0.2 ml of 40 per cent. ampholine solution. A volume of 2 to 3 ml of 0.5 per cent. orthophosphoric acid was added to the solution surrounding the anode. The contents of the isoelectric focusing columns were eluted and monitored as fractions by measuring the absorbance at a wavelength of 280 nm in a Unicam SP500 spectrophoto- meter. The fractions containing the individual separated proteins were pooled and chromato- graphed on a column of Amberlite IRC 50 CG Type I1 200 to 400-mesh ion-exchange resin of diameter 6 mm and height approximately 20 mm. The column was equilibrated against 0.01 M phosphate buffer at pH 6.97. The sugar was removed by washing the column with 3 ml of water.The protein and most of the ampholine were eluted with 1.0 M sodium chloride solution. The fractions collected from the IRC 50 column were monitored again for protein content by measurement of the absorbance at 280 nm and the appropriate fractions containing protein were pooled. This eluate of pooled fractions was added to a Sephadex G-25 column of height 20 cm and diameter 2.0 cm, which had previously been equilibrated against 0.01 M ammonium hydrogen carbonate solution. The protein was eluted with a solution of 0.01 M ammonium hydrogen carbonate and concentrated by freeze-drying for 16 hours. The proteins were hydrolysed in 6~ hydrochloric acid at 105 "C for 24 hours, after first removing air from the hydrolysis mixture by passing a rapid stream of nitrogen through it for 1 hour.13 The amino-acid compositions of the hydrolysates of the proteins were determined by means of an improved system of analysis described previously.9 Ribonuclease A was used as the test material.January, 19731 AMINO-ACID COMPOSITION OF PROTEINS TABLE I AMINO-ACID COMPOSITION OF BOVINE RIBONUCLEASE A, BATCH UK3, The experiments were carried out in the LKB 8100 electrofocusing column BEFORE AND AFTER ISOELECTRIC FOCUSING Percentage of total residues f A t 27 Amino compound Methionine sulphoxides Threonine .. .. Serine .. .. Glutamic acid . . Proline . . .. Glycine . . . . Alanine . . . . Cystine . . .. Valine .. .. Methionine . . .. Isoleucine . . .. Leucine . . .. Tyrosine . . .. Phenylalanine . . Lysine . . .. Histidine .. .. Arginine . . .. Oxidised cystine . . Oxidised methionine Total masslpg . . Isoelectric point . . Cysteic acid . . .. Aspartic acid . . .. .. .. . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Original protein 0.17 0.56 13.00 7.45 9-99 11.31 2.55 1.43 6.69 5-7 1 5.45 3-27 1-20 1.87 7.30 3.41 10.10 3.40 4-79 2.9 14.6 1459 - Protein treated once Peak I Peak I1 Peak I11 2.54 0.68 1.70 0.90 0.96 1.00 13.37 12.97 13.10 7.69 7.42 7.56 9.95 9-60 10.13 11-86 11-19 11.64 2.29 2.77 2.55 1.56 1.42 1.69 6-71 6.36 6-6 1 2.91 5.43 3.81 6-16 6.29 6-19 2.43 2.96 2.13 1-62 1.60 1-53 1.86 1.92 1.91 6.79 7.42 6-6 1 3.32 3.43 3.4 1 9.80 9-22 10.03 3-65 3-32 3.67 4.72 5-06 4.75 P 46.7 11.1 30.9 27.0 24.5 32-0 1772 402 1664 8-95 9.10 9.40 Protein treated twice, peak I1 1.00 1.43 13.17 7.43 9.84 11.62 2.58 1.41 6-88 5-05 6.21 1.76 1.62 1.93 6.83 3-45 10.07 3-06 4.48 16.5 44.8 558 9.10 Peak 11 in presence of ascorbic acid, treated once 0.17 0.39 12.96 7-02 9.37 11.38 2.47 1.33 6-30 5.9 1 6.12 3.36 1-61 1.81 7.28 3-46 9.73 3.58 5.09 2.8 10.3 6984 9.10 RESULTS The isoelectric focusing of a commercially available chromatographically pure sample of bovine ribonuclease A is illustrated in Table I.The amino-acid composition of the original sample, together with those of the separated fractions indicated by peaks I, I1 and I11 in Fig. 1, are shown. The protein contained in peak I1 was isolated by the normal LKB I "H0 Peak II PH 7.0 W c 2 L w 2 Fraction number (fraction volume, 1.0 ml) Fig. 1. Fractions of ribonuclease A isoelectrically focused in the absence of ascorbic acid.(LKB equipment)28 JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THE [Analyst, Vol. 98 procedure and freed from sucrose and ampholine, and a portion of the product was treated again in the isoelectric focusing column by the normal procedure with LKB equipment (Fig: 2). The amino-acid composition of the twice-treated protein is given in Table I. The original protein was isoelectrically focused in the presence of 10 ml of 10 per cent. ascorbic acid solution and only two fractions resulted (Fig. 3), viz., a main peak that corresponded to peak I1 and a very small peak that corresponded to peak I11 of the three peaks that resulted from the original protein sample. The amino-acid composition of the main peak obtained in the presence of ascorbic acid is shown in the final column in Table I.0.6 PH PH 10.0 7.0 0 Fraction number (fraction volume, 1.0 ml) Fig. 2. Fractions of bovine ribonuclease A obtained when the main component (peak 11, Fig. 1) is treated a second time by isoelectric focusing (LKB equipment) In Table I1 are shown the results of the analyses of the main peak (11) obtained by isoelectric focusing of bovine ribonuclease A in the presence of ascorbic acid (Fig. 3) or 2,2’-thiodiethanol as antioxidant (Fig. 4). The amino-acid composition of the original protein is also given in Table I1 for reference purposes. 2 .o 1.5 Q, t m -e 1.c a n a 0-E 0.I PH 10.0 Peak II ,u 10 20 30 40 50 60 3 Fraction number (fraction volume, 1.25 ml) Fig. 3. Fractions of ribonuclease A separated in the presence of ascorbic acid (LKB equipment)January, 19731 AMINO-ACID COMPOSITION OF PROTEINS TABLE I1 ANALYSES OF BOVINE RIBONUCLEASE A (PEAK 11) IN THE PRESENCE AND ABSENCE OF ANTIOXIDANT The LKB apparatus was used, and the results relate to batch UK3, except those in the final column, which relate to batch UK6 29 Amino compound Methionine sulphoxides Threonine .. .. Serine .. .. Glutamic acid . . Proline . . .. Glycine . . .. Alanine . . .. Cystine . . .. Valine .. .. Methionine . . .. Isoleucine . . .. Leucine . . .. Phenylalanine . . Histidine . . .. Oxidised cystine . . Oxidised methionine Cysteic acid . . .. Aspartic acid .. Tyrosine . . . . Lysine . . .. Arginine . . .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. . . . . .. . . Original protein 0.17 0.56 13.00 7.45 9-99 11.31 2-55 ' 1-43 6.69 5.7 1 5.45 3.27 1.20 1.87 7.30 3.41 10.10 3.40 4-79 2.9 14.6 Percentage of total residues A 1 In presence of I n absence of antioxidant antioxidant A I -I - 6 per cent.10 per cent. Treated Treated Ascorbic 2,2'-thiodi- 2,2'-thiodi- once twice acid ethanol ethanol 0.68 1.00 0.17 0.61 0-69 0.96 1.43 0.39 0.37 1.93 12.97 13.17 12.96 12.96 13.39 7-42 7.43 7.62 7.50 7.66 9-60 9-84 9-37 9-67 9.27 11.19 11.62 11-38 11.35 11.59 2.77 2.58 2.47 2.48 2.44 1.42 1.41 1.33 1.37 1-35 6-36 6.58 6.36 6.43 6.66 6.43 5.05 5.9 1 6.86 6.24 6.29 6-2 1 6-12 6.20 6.60 2-96 1-76 3.36 3.29 1-54 1.60 1.52 1.61 1-63 1.85 1-92 1.93 1.81 1.82 1.87 7.42 6-83 7.28 6.94 7-21 3-43 3.45 3.46 3-37 3.38 9.22 10.07 9.73 10.03 9-36 3.32 3.66 3.58 3.80 3.69 5.06 4.48 6-09 4.7 1 4.59 11.1 16-5 2.8 9.9 11-6 24-5 44.8 10.3 10.0 85.7 The results of the analyses of two batches of bovine ribonuclease A and the fractions resulting from them after isoelectric focusing on both the LKB and ISCO equipment are shown in Table I11 (see Figs.1 and 5). leak I I Peak Ill & 0*0' Ib i0 30 40 50 60 70 80 Fraction number (fraction volume, 1-1 ml) Fractions of bovine ribonuclease separated in the presence of 6 per cent. 2,2'-thiodiethanol solution (LKB equipment) Fig. 4. D30 O.OL 1'0 ;o 3b 40 El0 6L ;o sto do 1;o JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THE TABLE I11 ISOELECTRIC FOCUSING OF BATCHES OF BOVINE RIBONUCLEASE A [AnaZyst, Vol. 98 110 Amino compound Methionine sulphoxides Cysteic acid . . . . 0.5 Aspartic acid Threonine .. Serine . . Glutamic acid Proline . . Glycine . . Alanine . . Cystine . . Valine . . Methionine . . Isoleucine . . Leucine .. Tyrosine . . Phen ylalanine Lysine . . Histidine . . Arginine . . Oxidised cystine - .. .. .. .. .. . . . . . . . . .. .. .. .. .. .. .. .. Percentage of total residues A Batch UK3 in A r 7 LKB apparatus /-=-, ISCO apparatus Peak I1 Peak I1 ,-*-. without with ascorbic ascorbic Original acid acid . . 0.17 0.68 0.17 . . 0.56 0.96 0.39 . . 13.00 12.97 12.96 . . 7.45 7-42 7-62 . . 9-99 9.60 9.37 . . 11-31 11.19 11.38 . . 2-55 2.77 2.47 . . 1-43 1.42 1-33 . . 6-69 6.36 6.36 . . 6-71 6.43 6-91 . . 5.45 6.29 6.12 . . 3.27 2.96 3.36 . . 1.20 1.60 1.61 . . 1-87 1.92 1.81 . . 3-41 3.43 3-40 . . 10.10 9.22 9-73 . . 3.40 3-32 3.68 .. 4-79 6.06 6.09 . . 2.9 11.1 2.8 . . 7-30 7-43 7-28 Without ascorbic acid 2-93 0-28 13.16 7.70 9-96 11.52 2.78 1-48 6-69 2.84 6-90 3.46 1.42 1.97 6.98 3.38 9.91 3.39 4.18 50.7 With * ascorbic acid 0.43 0.84 13.28 7.65 9-83 11.36 2-69 1.40 6.43 6.58 6-70 3-18 1.31 1.86 7.20 3-34 10.06 3.46 4-61 6.9 Batch UK6 in ISCO apparatus* W l t h o u t h ascorbic ascorbic Original 0.63 0.63 13.15 7-47 9.41 11-28 2-03 1.37 6.39 6.63 6.98 3-29 1.69 1.93 7.42 3-42 10.23 3-65 4.48 10.1 acid 1.29 0.78 13.63 7.68 9.79 11.72 2.37 1.39 6-70 3.85 6-34 2.80 1.66 1-77 7-03 3.34 9.78 3.66 4.44 24.9 acid 1.40 0.13 13-49 7.62 9.64 11.81 2.55 1.40 6.77 3-86 6-52 3.63 1-81 1.78 6.87 3-29 9.72 3.62 4.30 27.7 * Column maintained a t 4 "C for the ISCO apparatus. The amino-acid compositions and isoelectric points of fractions of a glycoprotein and thyrotrophin isolated by the normal isoelectric focusing procedure with the LKB equipment are given in Table IV.1.5 Peak I I Fig. 5. Fractions of bovine ribonuclease A separated by the ISCO apparatus in the presence of ascorbic acidJanuary, 19731 AMINO-ACID COMPOSITION OF PROTEINS TABLE IV AMINO-ACID COMPOSITION OF PROTEINS AFTER NORMAL ISOELECTRIC FOCUSING I N THE LKB APPARATUS Percentage of total residues 31 Amino compound Cysteic acid . . .. Aspartic acid . . 0 . Methionine sulphoxides Threonine . . ,. Serine . . .. .. Glutamic acid . . .. Proline . . .. .. Glycine . . .. .. Alanine . . .. * . Cystine . . .. .. Valine . . . . .. Methionine . . .. Isoleucine . . * . Leucine * . .. Tyrosine .... Phenylalanine . . .. Lysine . . .. .. Arginine .. .. Glucosamine . . .. Galactosamine . . . . Histidine . . .. Oxidised cystine . . Isoelectric point . . .. .. .. .. .. .. .. .. .. .. .. .. . . . . .. .. .. .. .. .. .. .. .. a-Glycoprotein*/pmol - A B 0.017 0.016 Trace Trace 0.147 0.185 0.098 0.127 0.092 0.121 0.176 0.231 0.065 0.082 0.089 0.117 0.099 0-130 0.007 0.02 1 0.084 0-112 0.014 0.016 0.053 0.064 0.124 0.141 0.037 0.049 0.066 0.075 0.035 0.043 0.112 0.143 0.040 0.057 0.042 0.058 54.8 28.0 5-1 5.5 - - ceThyrotrophin/pmol 0.1831 0,0441 0.662 0.879 0.600 0-705 0.696 0-432 0.710 0,295 0.667 0-268 0.344 0,379 0.587 0.366 0.418 0.180 0.852 0.268 0.321 23.8 8.25-8.30 * Focused in 6 M urea solution after neuramidase treatment. The relative positions or p l values of bovine ribonuclease A fractions after separation by isoelectric focusing under different conditions are shown in Table V.TABLE V RELATIVE POSITIONS OF FRACTIONS FROM BOVINE RIBONUCLEASE A AFTER SEPARATION BY ISOELECTRIC FOCUSING Bovine ribonuclease system Fractions Isoelectric point Peak I 9.4 Peak I1 9-25 Peak I11 8.95 Without ascorbic acid . . . . .. .. With ascorbic acid . . . . . . Peak I1 9.15-9.20 Peak I11 8.95-9.00 Peak I 9-10 Peak I1 9.0 Peak I11 8.8 With 5 per cent. 2,2’-thiodiethanol . . . . Peak 11, treated twice without ascorbic acid Peak I Peak I1 9.1 5-9.2 0 9.06-9.10 Peak I - Peak I1 9.26 Peak I11 - \Vith 10 per cent. 2,2’-thiodiethanol . . . . DISCUSSION The heterogeneity of proteins found by other workersG8 after the treatment of the proteins by the normal isoelectric focusing procedure has been confirmed in the present study.When the solution in the column of sucrose density gradient containing the frac- tionated components of a protein has reached the stage of equilibrium, and is eluted directly and monitored for absorbance at 280 nm, the elution pattern indicates the individual com- ponents, In addition, the portions of solution finally eluted show absorbance values that32 [Analyst, vol. 98 increase steadily (see Figs. 1 to 5 ) to a maximum. These highly absorbing portions correspond to the layer of sucrose at the top of the sucrose gradient. It is common for a greyish film to be formed on the top of the sucrose gradient at the interface between the sucrose solution and the aqueous solution containing the phosphoric acid used to surround the anode.The high absorbance value at 280nm and the presence of trace amounts of insoluble material indicate the possibility of the presence of denatured protein at the interface between the sucrose column and the aqueous solution floating above the column. The analyses of the component fractions of proteins isolated by isoelectric focusing for amino-acids show that cysteic acid and methionine sulphoxides occur to an extent greater than in proteins isolated by chromatography, electrophoresis or ultrafiltration. The hydrolysates of cc-glycoprotein, thyrotrophin and bovine ribonuclease A have been examined in the present work by the improved system of automatic analyses for amino-acids described earlier.9 In a preliminary communication,14 it was shown that appreciable amounts of cystine and methionine were present in their oxidised states in the hydrolysates.Gordon and Louis15 examined an a, acute phase globulin of rats by the normal isoelectric focusing procedure and obtained two fractions. They reported that the isoelectric focusing led to alterations in at least some of the protein molecules. Bovine thyrotrophins have been separated by Fawcett, Dedman and Morrisls by using the normal isoelectric focusing procedure with the LKB 8100 apparatus. They reported the presence of four components, each with a different isoelectric point, and they gave the biological activities of three com- ponents. In a previous cornmunication,l4 the degree of oxidation of a thyrotrophin of iso- electric point 8-65 to 8-70 was reported as 13 per cent., but in the thyrotrophin of isoelectric point 8.25 to 8.30 reported in the present paper, the degree of oxidation of cystine was 23.8 per cent.It is possible that the electrolytic oxidation of a protein during isoelectric focusing may produce fractions with apparently specific isoelectric points. Each fraction recognised on the elution pattern after monitoring the eluate for absorbance a t 280nm may consist of a mixture of protein molecules, each of different charge, as monomers, dimers or polymers whose total charges may differ so little from each other that they cannot be resolved in the pH gradient. The experimental results obtained from the analyses of the bovine ribonuclease A frac- tions for amino-acids show that when the major fraction (peak 11) was isoelectrically focused a second time, the amount of the arginine residues in the protein thus isolated decreased from 5.06 to 4.48 per cent.(compared with 5-09 per cent. of arginine in peak I1 when the isoelectric focusing was performed in the presence of ascorbic acid as antioxidant). This degradation of arginine in the fraction in peak I1 (see Fig. 1) when submitted twice to iso- electric focusing might occur as the result of reduction of the original amino-acid during the electrochemical procedure. Arginine is known to be converted into ornithine by any one of four well known reaction^,^^,^^ and alkaline solutions may cause the conversion of arginine into ornithine. The system of chromatography used to examine the hydrolysates of proteins in this study would not resolve ornithine, if it was present, from lysine, and in the analysis of peak I1 obtained from bovine ribonuclease A subjected twice to isoelectric focusing (see Table I) the decrease in arginine was accompanied by an increase in the apparent amount of lysine (cf., results for lysine in Table I).It is possible that the enlarged peak that is normally occupied by lysine contained a small amount of ornithine produced by the electrolytic decomposition of a portion of the arginine residues in the bovine ribonuclease A molecules. The oxidation of cystine and methionine, which occurs when isoelectric focusing is used in the normal procedure described by the original authors,lP2 can be controlled by the use of a suitable antioxidant such as ascorbic acid.The bovine ribonuclease A used in these experiments was fractionated into three components when it was submitted to isoelectric focusing in the presence of 10 ml of either 5 per cent. (Fig. 4) or 10 per cent. thiodiethanol solution on the LKB column. Only two fractions of the protein were obtained when the ribonuclease A was examined in the presence of ascorbic acid (Fig. 3) with the LKB apparatus. The isolation of bovine ribonuclease A in the ISCO isoelectric focusing equipment from a commercial supply of the protein produced only two components, whereas three fractions were separated by the LKB equipment. The pH gradient used with the ISCO apparatus was produced by an ampholine mixture designed to yield a pH range of 8 to 10, extended by means of arginine, triethanolamine and diaminoethane to a pH of approximately 11.5.JACOBS: EFFECT OF ISOELECTRIC FOCUSING ON THEJanuary, 19731 AMINO-ACID COMPOSITION OF PROTEINS 33 The normal isoelectric focusing procedure adversely affected the tyrosine residues in the bovine ribonuclease A sample. The differences in the results for the percentage tyrosine residue values when the main component (peak 11) of bovine ribonuclease A was isolated in the presence of ascorbic acid and also after treatment once or twice by the normal isoelectric focusing procedure, are indicated in Table I. This effect on the percentage tyrosine residue value is shown also in Table 111, which includes results obtained by using both the LKB and ISCO isoelectric focusing equipment.The earlier observation^^^^^^ on the oxidation of the sulphur-containing amino-acids of a-glycoprotein and thyrotrophin have been supported by similar observations on the corre- sponding amino-acids of the commercial samples of bovine ribonuclease A, which was selected as a test material because of its relatively high concentration of sulphur-containing amino- acids. Provided that a suitable antioxidant is used to protect the sucrose density gradient, the protein can be isolated with minimum modification during the isoelectric focusing procedure, which takes approximately 16 hours. The author is indebted to Mr. R. A. Faulkes for skilled technical assistance, and thanks Dr. J. Fawcett and Dr. D. Leaback for the use of the LKB and ISCO isoelectric focusing columns, respectively. Loan of the ISCO apparatus from Shandon-Southern Instruments Ltd. is gratefully acknowledged. The author also acknowledges gifts of samples of ct-glyco- protein, for which he thanks Dr. A. H. Gordon, and of cc-thyrotrophin, for which he thanks Dr. Fawcett. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. REFERENCES Svensson, H., Acta Chem. Scand., 1961, 15, 326. Vesterberg, O., and Svensson, H., Ibid., 1966, 20, 820. Kolin, A., J . Chem. Phys., 1954, 22, 1628. Vesterberg, O., Biochim. Biophys. Acta, 1968, 168, 218. Quast, R., and Vesterberg, O., Acta Chern. Scand., 1968, 22, 1499. Lewis, U. J., Cheever, E. V., and Hopkins, W. C., Biochiwt. Biophys. Acta, 1970, 214, 4987. Norton, I. L., Pfunderer, P.. Stringer, C. D., and Hartman, F. C., Biochemistry, 1970, 9, 4952. Kaplan, L. J., and Foster, J. F., Ibid., 1971, 10, 630. Jacobs, S., Analyst, 1970, 95, 370. -, Chem. G. Ind., 1955, 944. -, Lab. Pract., 1963, 12, 557. -, Protides Biol. Fluids, 1964, 11, 463. -, Meth. Biochem. Analysis, 1966, 14, 177. -, Protides Biol. Fluids, 1971, 18, 499. Gordon, A. H., and Louis, L. N., Biochem. J., 1969, 113, 481. Fawcett, J. S., Dedman, M. L., and Morris, C. J . O., FEBS Lett., 1969, 3, 250. Schultze, E., and Winterstein, E., 2. physiol. Chem., 1898-1899, 26, 1. Greenstein, J. P,, and Winitz, M., Editors, “Chemistry of the Amino Acids,” Volume 3, John Wiley Received January 26212, 1972 Accepted JuZy loth, 1972 & Sons, New York, 1961, p. 2477.