ANALYTICAL BIOCHEMISTRY 141, 397-401 (1984)
`
`Sodium Sulfite as an Antioxidant in the Acid Hydrolysis
`of Bovine Pancreatic Ribonuclease A
`
`J. K. SWADESH, T. W. THANNHAUSER, AND H. A. SCHERAGA!
`
`Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853
`
`Received February 24, 1984
`
`Treatment of hydrochloric acid with sodium sulfite prior to the acid hydrolysis of bovine
`pancreatic ribonuclease A has been found to suppress the oxidation of cystine, methionine,
`and tyrosine without adversely affecting the recoveries of other aminoacids.Statistical analysis
`ofthe results indicated that the assumption of the independence of the mean and the variance,
`an assumption commonly used in the evaluation of the effects of various treatments, may not
`be valid in evaluating antioxidants used in the acid hydrolysis of proteins.
`KEY WORDs: aminoacid analysis; antioxidant; sodium sulfite; hydrochloric acid; hydrolysate.
`
`BGhlen and Schroeder (1) have noted the
`need for a simple procedure for the acid
`hydrolysis of proteins and peptides that does
`not require a separate determination oftryp-
`tophan, tyrosine, methionine, cysteine, and
`cystine. These authors used thioglycolic acid
`to prevent the oxidation of the first three of
`these amino acid residues, but required a
`separate analysis under oxidizing conditions
`for the determination of the sum of cysteine
`and cystine as cysteic acid. Inglis and Liu (2)
`used posthydrolytic conversion of cysteine,
`cystine, and cysteic acid to S-sulfocysteine as
`a strategy in amino acid analysis, and Inglis
`(3) has coupled this method with prehydro-
`lytic alkylation of cysteine as a procedure to
`differentiate between cystine and cysteine
`and to recover all amino acids,
`including
`tryptophan. Other authors (4-6) have used
`phenolas an antioxidant, and dimethylsulf-
`oxide has been used (7) to convert cysteine
`or cystine to cysteic acid during hydrolysis.
`Each of these methods has advantages and
`disadvantages.
`In particular, prehydrolytic
`derivatization of cysteine with 4-vinylpyridine
`or iodoacetate (3) requires the removal of
`the alkylating agent prior to hydrolysis, and
`
`'To whom request for reprints should be addressed.
`
`would therefore be difficult to apply to very
`large or very small samples or samples of
`low molecular weight. For reasons of conve-
`nience, hydrochloric acid is often preferred
`as the hydrolytic agent, but extensive oxida-
`tion is sometimes observed. The present work
`explores the use of sodium sulfite to improve
`the recoveries of cystine, methionine, and
`tyrosine in the acid hydrolysis of bovine
`pancreatic ribonuclease A, a protein that
`does not contain cysteine or tryptophan. The
`practical benefits conferred by pretreatment
`with sodium sulfite are discussed, and the
`general requirements fora statistical study of
`the effects of an antioxidant are explored.
`
`MATERIALS AND METHODS
`
`Sodium sulfite (Na2SO3) was purchased
`from Fisher Scientific Company. Bovine pan-
`creatic ribonuclease A, type I-AS, was pur-
`chased from Sigma, and was purified by ion-
`exchange chromatography on carboxymeth-
`ylcellulose (8) purchased from Whatman.
`Hydrochloric acid (12 M) was either pur-
`chased from Mallinckrodt and purified by
`simple or fractional distillation by a procedure
`designed to produce a constant-boiling frac-
`tion (9), or was purchased from Baker Chem-
`ical Company (Ultrex) and diluted to 6 M.
`
`_
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`-397-
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`398
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`SWADESH, THANNHAUSER, AND SCHERAGA
`
`The Mallinckrodt HCI (lot KMPE) exhibited
`an elevated boiling point during the latter
`part of the simple distillation, and complete
`destruction of cystine, methionine, and ty-
`rosine was observed with the use of this
`distilled acid in the absence of pretreatment
`with Na2SQ3.
`Eleven groups of replicate hydrolysates,
`each containing 4-6 samples, were prepared.
`The first four groups of replicates were pre-
`pared as 0.25-ml aliquots of simply or frac-
`tionally distilled Mallinckrodt acid which had
`been treated with | mg/ml of Na,SO;, or
`not treated, and into which ribonuclease A
`had been dissolved at a concentration of 1
`mg/ml. The fifth group of replicates was
`prepared as 10-ul aliquots of fractionally
`distilled Mallinckrodt acid containing protein
`at a concentration of 27 mg/ml. The next
`two groupsof replicates were prepared as 10-
`ul aliquots of Ultrex acid which had been
`treated with 7 mg/ml of Na2SO;, or not
`treated, and into which the protein had been
`dissolved at a concentration of 10 mg/ml.
`The final four groups of replicates were pre-
`pared as 10-ul aliquots of Ultrex acid with a
`protein concentration of 10 mg/ml. In two
`of these groups of replicates,
`the acid had
`been purged for 1 h with helium gas prior to
`sample preparation and, to two of the groups
`of replicates, 10 mg/ml of Na2SO; had been
`added to the HCI prior to the addition of
`protein.
`Hydrolyses were performed in 8-mm-i.d.
`glass tubes. The solutions were degassed by
`the freeze-pump-thaw method (10) at 0.03
`mm Hg. The freeze-pump-thaw cycle was
`repeated three times. The additional precau-
`tion of decreasing the pressure in the tube to
`about 2 mm Hgpriorto freezing the sample
`in liquid nitrogen was observed for the final
`four groups of replicates; this should prevent
`the condensation of oxygen. All samples were
`hydrolyzed at 110°C for 24 h, and the HC)
`was removed under vacuum. Samples were
`analyzed on a Technicon TSM amino acid
`autoanalyzer.
`
`In the following section, groups ofrepli-
`cates have been labeled with the symbols M
`or U to indicate Mallinckrodt or Ultrex acid,
`with the symbols s or f to indicate that the
`HCl was distilled by simple or fractional
`distillation, and with the symbol He if the
`HCl had been purged with helium. The
`symbol ¢ indicates that no sulfite was added,
`and the symbol S indicates the addition of
`Na,SO;3.
`Statistical analysis was performed according
`to the technique of variance analysis (11).
`This technique apportions the variance that
`exists between groupsofreplicates to random
`error or to the effects of the various treat-
`ments, i.e., to the method ofdistillation, to
`the effect of purging with helium, or to the
`effect of addition of Na,SO3. In the technique
`of variance analysis, the statistical significance
`of the effect of a particular treatment
`is
`determined by an F test. To evaluate the
`effects of various treatments in the first four
`groupsof replicates, a two-sided F test was
`used,
`i.e.,
`it was assumed that addition of
`Na2SO;could either increase or decrease the
`yields of the oxidizable residues. The results
`demonstrated unambiguously that Na2SO;
`does not decrease the yields; hence, one-sided
`F tests were used in all subsequent analyses
`of variance. One cannot combine groups of
`replicates for the purpose of variance analysis
`unless it is known that the mean and the
`variance are approximately independent of
`one another. For the purpose of investigating
`the dependence of the variance on the mean,
`a phenomenon formally known as hetero-
`scedasticity, Bartlett’s test (11) was used. A
`simpler, but qualitative technique involves
`graphing the variance or standard deviation
`against the mean.
`
`RESULTS AND DISCUSSION
`
`The principal results of this work are pre-
`sented in Table 1. Under conditions of high
`sample dilution, extensive destruction of the
`oxidizable residues occurred in the absence
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`SODIUM SULFITE AS ANTIOXIDANT IN RIBONUCLEASE A ACID HYDROLYSIS
`
`399
`
`HYDROLYSES OF BOVINE PANCREATIC RIBONUCLEASE IN MALLINCKRODT HCl
`
`No Na,SO, added
`
`Na,SQ, added
`
`TABLE |
`
`Replicate group Replicate group§Number ofNumber
`
`
`Method of
`Amino acid
`number and
`of residues
`number and
`residues
`treatment
`residue
`mnemonic*
`recovered’
`mnemonic
`recovered
`
`Theoretical
`
`Simply
`distilled HCI
`
`Fractionally
`distilled HC
`
`Half-Cys
`Met
`Tyr
`
`Half-Cys
`Met
`Tyr
`
`8
`4
`6
`
`8
`4
`6
`
`| Ms
`
`3 MsS
`
`0.0 (—-)
`0.0 (—)
`0.0 (—)
`
`2 Mf¢é
`
`4 Mfs
`
`1.48 (1.28)
`1.59 (0.96)
`1.76 (0.83)
`
`4.84 (0.51)
`3.25 (0.45)
`1.88 (1.50)
`
`5.07 (0.19)
`3.56 (0.18)
`3.31 (4.31)
`
`* See text for the nomenclature of the replicate groups. Each group contained five replicates. All samples were
`prepared with a protein-to-HC! molar ratio of 1:80,000. In samples MsS and MfS,the protein-to-Na,SO, molarratio
`was 1:100. In a fifth group of four replicates, the protein-to-HCl molar ratio was increased to 1:3,000, but no sulfite
`was added. The observed recoveries were: Cys 5.45 (0 = 0.97), Met 3.86 (o = 0.12), and Tyr 4.61 (a = 0.42)residues.
`’ Values reported are the number of residues recovered, based on an assumed recovery of Ala plus Glu equal to
`24, Values in parentheses are standard deviations.
`
`of Na,SO3. In a footnote to Table 1, it is
`sided F test on groups 6 and 7 demonstrated
`that
`the addition of sulfite improved the
`noted that, at a higher concentration of pro-
`tein, fair recoveries of the oxidizable residues
`recovery of methionine at a confidencelevel
`were observed. Hence, although the quality
`of 99.9%, and the recovery of cystine at a
`of the HCl as purchased was undoubtedly
`confidence level of 97.5%. The confidence
`poor, perhaps due to the length of time
`level on the increased recovery of tyrosine is
`between production and purchase, it is evi-
`below 90%.
`Two-factor analysis of the variance of
`dentthat recoveries of the oxidizable residues
`are improved by distillation of the acid, by
`groups 8-11 was not conclusive because of
`reduction of the ratio of HCI to protein, and
`the limiting effect of the protein composition;
`by the addition of Na,SO;. Two-factor anal-
`as one approaches quantitative yield in the
`ysis of the variance, using a two-sidedFtest
`absence of any treatment, increases in per-
`as described in the previous section, showed
`centage yield due to the effects of the various
`that addition of Na2SO; increased the recov-
`treatments necessarily become small. The
`ery of the oxidizable residues with a confi-
`improvements of the yields of cystine and
`dence level greater than 99%. Fractional dis-
`methionine, attributable either to purging of
`tillation of the HC] also improved the re-
`the HCI with helium or to the addition of
`coveries with a confidence level greater
`Na2SO;, are not statistically significant, al-
`than 97.5%.
`though it is germaneto note that addition of
`Additional tests, using a higher grade of
`Na,SO, qualitatively seems to improve the
`acid, were also performed, and these results
`yields of these residues. For tyrosine, however,
`are contained in Table 2. Groups 6 and 7
`a one-sided F test
`indicated at
`the 90%
`confidence level that the recovery is increased
`could not be combined with the remaining
`groups because of heteroscedasticity. A one-
`by purging with helium, by addition of
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`SWADESH, THANNHAUSER, AND SCHERAGA
`
`TABLE 2
`
`HYDROLYSES OF BOVINE PANCREATIC RIBONUCLEASE A IN ULTREX ACID
`
`Number of
`Number of
`Number of
`tyrosine
`methionine
`half-cystine
`residues
`residues
`residues
`Replicate group§Number of
`(theory = 8)
`(theory = 4)
`(theory = 6)
`number and
`replicates
`Molar ratio of
`mnemonic*
`in group
`protein:HCl:Na-SO,
`
`Mean
`
`SD
`
`Mean
`
`SD
`
`Mean
`
`SD
`
`6 U¢'
`7 US’
`8 Ud
`9US
`10 U¢-He
`11 US-He
`
`6
`6
`5
`5
`5
`5
`
`1:6800:0
`1:8200:80
`1:5400:0
`1:5400:72
`1;5400:0
`1:5400;72
`
`6.26
`7.12
`7.57
`7.64
`7.68
`7.83
`
`0.99
`0.40
`0.41
`0.21
`0.21
`0.19
`
`3.61
`3.76
`4.02
`4.00
`4.04
`4.14
`
`0.03
`0.14
`0.16
`0.14
`0.13
`0.06
`
`4.61
`5.05
`5.22
`5.40
`5.48
`5.70
`
`0.12
`0.09
`0.22
`0.27
`0.16
`0.30
`
`@ See text for nomenclature. U¢d' and US’differ from U¢ and US, respectively, because, in the former, the pressure
`inside the hydrolysis tubes was not reduced prior to freezing the sample in the freeze-pump-thaw cycle.Statistical
`analysis, described in the text, precludes the combination of Ud¢’ with U¢ or US’ with US.
`
`Na2SO3, and by a synergistic interaction of
`these treatments. Additional experiments, not
`presented in this paper, demonstrated that
`the addition of up to 40 mg/ml of Na2SO;
`to the hydrolysis acid did not reduce the
`yields of the other amino acids of ribonu-
`clease.
`We have not been able to establish the
`mechanism by which pretreatment of HCl
`used in acid hydrolysis improves the yields
`of cystine, methionine, and tyrosine. In acidic
`solution, Na2SO;
`is converted to SO, gas,
`most of which is slowly evolved from solution
`prior to addition of the sample, and the
`remainder of which is presumably removed
`as the pressure in the hydrolysis tube is
`reduced under vacuum. The apparent syn-
`ergism of purging with helium and adding
`Na,SO3, observed in the recoveries of tyrosine
`in groups 8-11, suggests that Na,SO; reduces
`trace oxidants present even in freshly prepared
`acid, and does not simply purge the HCI of
`dissolved molecular oxygen. Oneinteresting
`observation, perhaps relevant to the elucida-
`tion of the mechanism by which antioxidants
`function, is the linear relationship obtained
`if the mean recovery of a given amino acid
`in the absence of Na2SO;is plotted against
`
`the mean recovery in the presence ofsulfite,
`i.e., if the result of Ms@ is plotted against
`that of MsS, that of Mf¢ against that of MfS,
`and so on. Hence, although we do not know
`the antioxidative mechanism,
`the effect of
`addition of Na2SO;3appears to be regular and
`predictable.
`Successful amino acid analysis depends on
`many factors, including the composition of
`the protein, the molar ratio of HCI to protein,
`the freshness of the hydrolysis acid used, and
`the experimental details of sample prepara-
`tion, The present work demonstrates that
`improved yields of the oxidizable amino acids
`can be obtained by pretreatment of the HCl
`with sodium sulfite. The practical applications
`of this observation are many. First, in some
`applications, it may be possible to substitute
`a lower grade of hydrolysis acid. Second,it
`may be possible to extend the effective shelf
`life of hydrochloric acid used in amino acid
`analysis. Third, Naz,SO3 may serve as a re-
`placement for phenol, which is difficult to
`purify. Finally, as amino acid analysis is
`extended to samples of smaller size, reduction
`of the volume of hydrolysis acid becomes
`increasingly difficult; hence, the use of an
`antioxidant becomes necessary. In many ap-
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`SODIUM SULFITE AS ANTIOXIDANT IN RIBONUCLEASE A ACID HYDROLYSIS
`
`401
`
`3.
`
`J.
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`. Skoog, D. A., and West, D, M. (1976) Fundamentals
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`
`plications, Na2SO; may prove to be the an-
`tioxidant of choice.
`
`ACKNOWLEDGMENTS
`
`This work was supported by research grants from the
`National Institute of General Medical Sciences (GM-
`14312), and from the National Science Foundation
`(PCM79-20279). JKS was an NIH Postdoctoral Fellow
`(1982-1984), We thank M. E. Denton, Y. Konishi, and
`C. A. McWherter for valuable discussions, and G. T.
`Montelione, S$. H. Lin, and E. A. Czurylo for helpful
`comments on the manuscript.
`
`REFERENCES
`
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`Inglis, A. S., and Liu, T. Y. (1970) J. Biol, Chem.
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