hydrolysis of proteins by microwave energydownloads.hindawi.com/journals/jamc/1991/745937.pdf · S. A.Margoliset al.Thehydrolysis ofproteins bymicrowaveenergy Resultsanddiscussion

Journal of Automatic Chemistry, Vol. 13, No. 3 (May-June 1991), pp. 93-95

The hydrolysis of proteins by microwaveenergy

Sam A. Margolis, Lois Jassie and H. M. KingstonNational Institute ofStandards and Technology, Centerfor Analytical Chemistry,Organic Analytical Research Division, Gaithersburg, Maryland 20899, USA

Microwave energy, at manually-adjusted, partial power settingshas been used to hydrolyse bovine serum albumin at 125C.Hydrolysis was complete within 2 h, except for valine andisoleucine which were completely liberated within 4 h. The amino-acid destruction was less than that observed at similar hydrolysisconditions with other methods and complete hydrolysis was achievedmore rapidly. These results provide a basis for automating theprocess of amino-acid hydrolysis.

Introduction

Microwave energy comprise waves of mm to 100 m andis found between the radio and the infra-red regions of theelectromagnetic spectrum. In aqueous or dielectricmedia, the microwaves may induce polarization insusceptible molecules, as well as promote the rotation oftrue molecular dipoles at rates approaching 2"45 x109 MHz, the frequency of the microwave commonlyused for commercial microwave ovens. If the dielectricrelaxation of the material is the same as the frequency ofthe radiation (109 to 1012 cycles/s), then the microwaveradiation will couple directly with the oscillating particlesin solution. The heat produced by microwave irradiationarises from two effects: (1) the frictional forces whichresult as the molecular dipoles align themselves with thepassing wave front; and (2) the movement of hydratedcharged ions or molecules with a dipolar solvent shelltoward their oppositely charged poles.

Amino-acids, peptides and proteins are charged mole-cules which have dipolar properties. Dipole moments ofthe ethyl esters ofselected amino-acids are approximately2" Debyes, and those ofselected amides range from 3"5 to5 Debyes [1]. A protein is composed of a number ofdipolar moieties which contribute to the net dipolemoment of the total protein. This is further modified bythe hydration shell of the molecule. It is conceivable thatthe electromagnetic energy could increase the rotationalforce on bonds connecting dipolar moieties to adjacentatoms, thereby reducing the energy required to breakbonds and increasing the probability for the occurrence ofsuch processes as hydrolysis.

The production of heat by microwave radiation(2450 MHz) ofelectrolyte solutions, such as concentratedHC1, which contain small amounts oforganic materials isalmost exclusively the result of the action of themicrowave on the major electrolytes and not on theorganic material. The combination of the heating effectand the possible increase in rotational forces on the

peptide bonds may catalyse more effectively the hydroly-sis of the peptide bonds and preserve the integrity of theless stable amino-acids.

The hydrolysis of proteins and peptides by microwaveenergy has been demonstrated under conditions wherethe microwave power was not controlled [2,3]. The heat-sensitive amino-acids, such as serine, threonine andtyrosine, showed a significant degradation (20, 40 and35% respectively) [2], which is characteristic for expo-sure of these amino-acids to temperatures between 172and 174 C. The objective of this study is to evaluate theutility of using controlled microwave power as a procedurefor hydrolysing proteins to their constituent amino-acids.

Methods

Reagents

The bovine serum albumin (BSA) used in this study wasNIST Standard Reference Material (SRM) 926 [4].High-purity water was prepared from distilled deionizedwater by subboiling distillation in a quartz apparatus. Ina similar quartz still, 10 mol/1 HC1 was prepared bysubboiling distillation of concentrated reagent gradeHC1. Amino-acid calibration standards were obtainedfrom Pierce (Rockford, Illinois). 9-Fluorenylmethoxy-carbonylchloride (FMOC) was obtained from Fluka(Ronkonkoma, New York, USA).

Duplicate BSA samples were prepared by adding 4"0 mlof an aqueous solution of albumin SRM 926 (16"05 mg/ml) to a clean acid-leached Teflon (PFA) pressure vesselwhich was free of metal impurities. Prior to the additionof the albumin solution, 1"5 ml of water and 6 ml of10 mol/1 HC1 were added to the vessel. The vessels wereflushed with nitrogen, tightly sealed and placed on acarousel in a laboratory microwave system [5]. Onevessel in each experiment was fitted with a pressuresensor and a fibre-optic thermometer suitable for use inmicrowave instruments [5]. Temperature and pressureequilibration was achieved in 5 min and was constantlymonitored with microwave power (145-98 W) beingadjusted manually. The pressure varied between 0"75and 0"96 atm (97 kPa), and the temperature between124"6 and 125"2 C.

Amino-acid analysis was performed and the amino-acidderivatives were prepared as described by Cunico et al.[6], except that an aliquot of base (1"2 mol/1NaOH) wasadded to an aliquot ofeither a standard mixture ofamino-acids or a protein hydrolysate to neutralize the HC1. TheFMOC amino-acids were analysed on a single pumpliquid chromatograph with low pressure gradient mixingand measured with fluorescence spectrophotometer.

Copyright U.S. Government 199193

S. A. Margolis et al. The hydrolysis of proteins by microwave energy

Results and discussion

The purpose of this study was to demonstrate thatproteins can be hydrolysed by microwave radiation asrapidly, or more rapidly, than by classical methods [7],and with less destruction of the labile amino-acids, suchas serine, threonine, and tyrosine. The results in table

Table 1. Effect ofmicrowave hydrolysis duration and amino-acidstandard on the amino-acid composition ofbovine serum albumin.

Amino-acid

Number of residues*Hydrolysis time2 h 4 h Theory

Arg 23 23 23Ser 31 28 28Asp 55 54 54Glu 79 80 79Thr 33 32 34Gly 17 18 16Ala 46 47 46Pro 30 29 28Met 4 4 4Val 30 34 36Phe 25 26 27Cystine 14 16 17Ile 11 13 14Leu 59 62 61His 18 19 17Lys 58 59 59Tyr 20 19 20

* Each value represents the mean value of the analysis oftwo separate hydrolysates calibrated against each of twoindependent standard amino-acid mixtures (a total offour measurements for each amino-acid at each timeperiod).

demonstrate that hydrolysis by microwave energy iscapable of achieving this objective. Bovine serum albu-min SRM 926 was selected as a substrate for thesestudies, because its sequence is known [8-10], andbecause its purity and homogeneity have been wellcharacterized for the purpose of using it as a clinicalreference material for measuring serum proteins. Thetemperature of 125C was selected for microwavehydrolysis in order to permit the completion of thehydrolysis step within 4 h and to be able to compare theseresults with those obtained at 2 and 4 h at 145 C [7].

Chen et al. [2] and Chiou et al. [3] did not regulate themicrowave power in their studies and this led todegradation of the thermally labile amino-acids. In thisstudy, the microwave power was manually regulated tomaintain the temperature at 125 + 2"1 C based upontemperature measurements during the hydrolysis pro-cess. The control of the temperature in these experimentsdepends on reaching an energy equilibrium betweenapplied microwave energy and heat loss from the samplevessel [5]. This form of control was not adequate toprovide unattended operation of the system. Becausesome of the amino-acid residues are susceptible todegradation, manual adjustments were necessary toprevent temperature drift. Fully automated temperaturefeedback control has been implemented on a similar

94

microwave unit to provide automated control for this typeof biochemical hydrolysis in the future 11 ].

A comparison of the amino-acid composition of thesample hydrolysed for 2 h to that hydrolysed for 4 hindicates the hydrolysis was complete within the 2 hperiod, with the exception ofa small percentage ofvaline,isoleucine and leucine residues the peptide bonds ofwhich are resistant to hydrolysis [7]. Tryptophan was notmeasured because it is not stable to acid hydrolysis.Threonine, serine and tyrosine, which are partiallydegraded by other hydrolysis procedures [5,6,11,12],appeared to be stable. This stability was confirmed byparallel exposure of amino-acid mixtures to microwaveradiation. These results indicate that microwave hydroly-sis is less destructive than the classical hydrolysisprocedure [7] and achieves complete hydrolysis at alower temperature, in a given period of time. Theseresults are comparable in precision and accuracy to thosereported by other investigators using other methods ofhydrolysis [6,7,12,13].

By the process of manual adjustment it is possible tocontrol the microwave power on the basis of the observedtemperature and thereby obtain reproducible hydrolysisconditions. This technique also provides the basis fordesigning a procedure for automating the process ofprotein hydrolysis and transferring this methodology togeneral laboratory use.

These results indicate for the first time that completehydrolysis of proteins can be achieved by exposure tomicrowaves under controlled conditions. The hydrolysiscan be carried out in a short time (2-4 h) at a relativelylow temperature (125 C). It appears that, in addition tothe thermal effect, microwave radiation may induce orfacilitate rupture of the peptide bond by a localizedinteraction with each polarizable amide group of thepeptide chain.

Note

Identification of any commercial products or equipmentdoes not imply recommendation or endorsement by theNational Institute of Standards and Technology, nordoes it imply that the material or equipment identified isnecessarily the best for the purpose.

References

1. WEAST, R. C., Handbook of Chemistry and Physics (TheChemical Rubber Co., Cleveland, 1972), p. E-54.

2. CI-IFN, S., CI-IlOV, S., YFN, Y. and WAN, K., InternationalJournal of Peptide and Protein Research, 30 (1987), 572.

3. CHIOU, S. and WANG, K., Journal of Chromatography, 491(1989), 424.

4. RFXDER, D. J. and SCI-IAFFER, R., In Standard ReferenceMaterials: Summary ofthe Clinical Laboratory Standards Issued bythe National Bureau OfStandards, edited by Seward, R. W. andMavrodineanu, R. (U.S. Government Printing Office,Washington, D.C., 1981), NBS Special Publication 260-71.

S. A. Margolis et al. The hydrolysis of proteins by microwave energy

5. KNasa’oN, H. M. and JASSIE, L. B., Introduction to MicrowaveSample Preparation: Theory and Practice (American ChemicalSociety: Washington, D.C., 1988), Chapter 6.

6. CuNICO, R., MAYER, A. G., Wv.I-IR, T. and SHF.FIjAn, T. L.,Biochromatography, 1 (1986), 6.

7. Roncn, D. and GigI, C. W.,Journal ofChromatography, 52(1970), 393.

8. PEa’gs, T., Clinical Chemistry, 23 (1977), 5.

9. WIEJERS, R. N. M., Clinical Chemistry, 23 (1977), 1362.10. Uwno, A., HoN, Y. M., AAXAK, N. and TAIV.DA, Y.,

Journal of Biochemistry (Tokyo), 98 (1985), 269.11. Knesa’oy, H. M. and WA.a’v., P. J., in preparation.12. MvJ.a’zv.R, N. M., ToJs, G. I., GvBF, S. and Srv.In, S.,

Analytical Biochemistry, 160 (1987), 356.13. SMxson, R.J., NwUBv.F, M. R. and LxJ., T. Y.,Journal of

Biological Chemistry, 251 (1976), 1936.

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hydrolysis of proteins by microwave energydownloads.hindawi.com/journals/jamc/1991/745937.pdf · S. A.Margoliset al.Thehydrolysis ofproteins bymicrowaveenergy Resultsanddiscussion