An Infrared Spectroscopic Study of the Hydrogen- Deuterium Exchange of Bovine Submaxillary Mucin*

Frank S. Parked and Martin H. Stryker* Department of Biochemistry, New York Medical College, New York, New York 10029

(Received 6 January 1969; revision received 7 February 1969)

An infrared spectroscopic method was used to s tudy the hydrogen-deuter ium exchange of a glycoprotein, bovine submaxillary mucin (BSM), dissolved in D~O. The pH-dependence of the rate and extent of the H - D exchange of BSS'[ was determined. The rate constant of the exchange decreased as pK increased from 3.7 to 5.3 and remained constant, at a minimum value, (0.82 =t=0.09) X 10 -2 min -~, from pH 5.3 to 7.2. The extent of the exchange decreased with increasing pH in the pH range 3.7 to 5.3 and levelled off from pH 5.3 to 7.2. I t is suggested tha t these results are due to a pH-dependent conformational change.

INDEX HEADINGS: Hydrogen-I)euter ium exchange; Glycoprotein.

INTRODUCTION

Bovine submaxillary mucin (BSM) is a member of a class of compounds, the salivary glycoproteins, which has been intensively studied in the laboratories of Pigman ~ and Got tschalk? The molecule, with a molecular weight of 4X 106, ~ consists of a long protein chain with numerous disaccharide and oligosaccharide side chains. 4,5 The disaeeharide side chains, composed of a hexosamine residue and a sialic acid residue, are a t tached by O-glycosidic linkages to the hydroxyl groups of serine and threonine. 5-8

From results obtained by infrared spectroscopy 9 and light-scattering ~ it has been suggested tha t the protein core is in a ~-configuration, or other extended form, and not an ca-helix. I t has also been found by light- scattering tha t at low ionic strength the molecule is a rigid rod, but when the ionic strength is increased to suppress the dissociation of the sialic acid earboxyl groups the molecule becomes more of a random coilJ °

A fuller understanding of the physiological functions of the mucins, which include protect ion of the tissues of the oral-gastro-intestinal tract, may come from a more complete knowledge of the conformation in solution of these molecules. Hydrogen exchange was therefore used to obtain information on the conforma- tion in solution of BSM and the effect of pH on the conformation. The l i terature on hydrogen exchange and its application to the s tudy of protein conforma- tions has been ably reviewed recently by Hvid t and Nielsen, ~t Harr ington et al . , a and Englander. 'a

* This research was supported by U. S. Public Heal th Service Research Gran t 5 RO1 NB 07625-01-02 from the National Ins t i tu te of Neurological Diseases and Blindness. The material in this paper is taken in par t from a thesis submit ted by Mar t in H. Stryker in partial fulfillment of the requirements for the degree of M.S. ir~ Biochemistry at New York Medical College. A preliminary report of this research was given at the Seventh National Meeting of the Society for Applied Spectroscopy, Chicago, Ill., 16 May 1968.

t Career Scientist of the Health Research Council of the City of New York under contract 1-323.

++ National Science Foundation Graduate Trainee, 1966-1967.

I. EXPERIMENTAL

A. Preparation of Bovine Submaxillary Mucin

BSM was extracted and isolated from frozen bovine submaxillary glands by the method described by Te t t aman t i and Pigman24 For these studies the total mucin was used, including both the major and minor components.

B. Hydrogen-Deuterium Exchange

The infrared spectroscopic method used was essen- tially tha t described by Blout et al. 15 and Hvidt . 16 The ins t rument used was a Perk in-Elmer model 521 grating infrared spectrophotometer . I t was calibrated against specific absorption bands of indene, poly- styrene, and atmospheric water. Perk in-Elmer fixed path length optical cells with CaF2 windows and a 0.1-mm optical pa th length were used.

To prepare for exchange studies a BSM stock solu- tion of known concentrat ion (approximately 1%) and pH in glass-distilled H20 was prepared. The pH of this stock solution was measured with a Radiometer model 25 pH meter equipped with a Radiometer G K 2 0 2 1 B combination electrode and standardized with Radiometer phosphate buffer. The pH values used throughout this work are the pH values of the stock solutions. No a t t empt was made to measure pD. (For a more detailed description of experimental procedure see Di Sabato and Ottesen. 17)

Portions of the stock solutions were lyophilized and then brought back to their original volumes with D20 (Bio-Rad Laboratories, 99.84 mole % D20). The spectrum of each of these samples was scanned between 1800 and 1400 cm -1 with pure D20 in the reference beam (Fig. 1). The t ime interval to scan this region was about 3 min. The time recorded for each scan was the t ime from the moment D20 was added to the lyophilized sample until the amide I I band at 1540 cm -1 was scanned. Room tempera ture ranged from 20 ° to 23°C but was usually constant on

Volume 23, Number 3, 1969 APPLIED SPECTROSCOPY 245

4 0

.600

- I ' I ' 1 ' I ' I ̧ -

":C ~ . . ~ - .

~.." :v--:~

";1 /,.. / '

20 I I I ~ I I I I I r800 1700 1600 1500 1400

C M - I

FiG. 1. Spectra of 1.37% BSM in I)20, pH of stock solution 3.91, after various times of exchange: (---) 6 rain; ( . . . ) 13 rain; ( ) 40 rain; (--.) 220 rain; (-..) heated for 4 h at 60°C.

any one day. Scanning was continued for 24 h a t various intervals.

A port ion of the sample was heated a t 60 ° for 4 h to produce a completely deutera ted sample. The heated solution was injected into a CaF~ cell and af ter reaching room tempera tu re it was scanned in place of the original sample.

The absorbance of the amide I band (Aamide I) was measured a t 1630 cm -~ with the absorbance at 1760 em -1 as the baseline. AamideI was found to be constant within a small experimental error. The absorbance of the amide I I band (Aamide II) ~VaS mea- sured at 1540 cm -~ as the difference in absorbanee between tha t of each scan and tha t of the complete ly deutera ted sample (Fig. 1).

The ratio (Aamide II/Aamide I) WaS used as a measure of the exchange. 15 (Aamide II/Aamide I) WaS plot ted vs t ime (Fig. 2). The curves thus obtained were extrapo- lated to zero t ime on semi-log paper. ~s The value of the absorbance ratio (Aamide II/Aamide I) at zero t ime is the value for the completely undeute ra ted sample. The percentage of unexchanged peptide hydrogen a toms u a t any t ime t is calculated f rom the following equat ion :

(A~miae H/A~,,,ia~ I), u = X 100. (1)

(Aami,te II/Aamide I)0

5 T E

%4 X

.500

~ . 4 0 0

3 0 0

~, .200 <

pH 7.10

~ . ~ _ ~ ~ ,.,----~-L.g'o. oH~.~-

0 i 2 5 4 5 TIME ( HOURS }

.FIG. 2. (Aamlde II/Aamlde I)t for BSM in D:O at several pH values: [] 7.10; O 4.81; • 4.25; A.3.78.

The hydrogen-deu te r ium exchange of a sialic acid, N-ace ty lneuraminic acid (General Biochemicals), was also studied. This was done to see whether there would be any contr ibut ion f rom the ca rbohydra te prosthet ic groups to the slow H - D exchange of BSh/I.

I I . R E S U L T S

The hydrogen-deu te r ium exchange experiments on N-ace ty lneuraminic acid showed very rapid exchange of the secondary amide hydrogen. The exchange was completed within seven minutes, i f these hydrogens behave in the same way when they are pa r t of the ca rbohydra te prosthet ic groups of BSM, then they do not interfere with measurements of the exchange of the pept ide hydrogens.

For each hydrogen-deuter ium exchange run with BSh~[ an initial first-order ra te constant of the ex- change k was calculated. The exchange was found to be first-order for an initial period of 20 to 35 min. Ra te constants were computed f rom a graph of

(A amiae II/A amide I) 0 In vs time.

(Aamide I I / A a m i d e I) t

The pH-dependence of this first-order ra te cons tant of the exchange is shown in Fig. 3. The ra te cons tant decreases with increasing p H until a min imum value is reached f rom p H 5.3 to 7.2. The average value of k in this p H range was found to be (0.82 ± 0.09) × 10 -2 rain -1.

The value of the ratio (Aamide II/Aamide I)0 WaS found by extrapolat ion for each of the runs in the p H range

2

O

i

~oo

t-- 8o

i -

0_

i i i

°a 4 ; 6 7 pH OF STOCK SOLUTION

FIG. 3. pH-dependence of first-order rate constant of H - D ex- change of BSM.

246 V o l u m e 23 , N u m b e r 3 , 1 9 6 9

7 0

0 0

0

0 0 6 0

5O

8 0

4 0

50

2 0

I0

O I

pH OF STOCK SOLUTION

Fro. 4. pH-dependence of extent of H-D exchange of BSM.

of minimum exchange rate. The average of these values was 0.48. This value is in close agreement with results reported by Blout et al. ~5 and by Hvidt. 16

The percentage of unexchanged peptide hydrogens u was calculated for each run using Eq. (1) with t = 5 h . 5~[easurable exchange had ended in all cases before 5 h. For those runs below pH 5.3 the relatively rapid rate of exchange made the extrapolation procedure in- accurate. For this reason, when using Eq. (1) on these r u n s (AamideiI/Aamide I)0 WaS taken as 0.48. For p i t values above 5.3 the individual extrapolated value for each run was used.

Figure 4 shows the variation of u with pH. There is an increase in u with increasing pH in the pH range 3.7 to 5.3, and then a levelling off from pH 5.3 to 7.2.

Ill. DISCUSSION

Most hydrogen exchange studies on proteins which have been done in sufficient detail can be interpreted by a model proposed by LinderstrOm-LangY This model assumes that a stable conformation (A1) is in equilibrium with one or more other conformations (A2) and that only the latter undergo exchange.

Ai~ -~- A2 "-+ exchange. (2)

Each amide hydrogen is assumed to exchange with a

rate constant k3 which is that found for structureless peptides under the same experimental conditions. Bryan and Nielsen 19 determined the dependence of k3 on pH and temperature for poly-DL-alanine. They found that it follows the equation:

ka = 50 (10-P~-tr 10~H--6) 10°'°5 (T-2°)min-i, (3)

where T is in °C. This relationship has been generally used to find k3 for any specific set of reaction condi- tions. Experimental hydrogen exchange rates are then a function of kl and k2, or the motility of the protein. With the effect of pH on k3 known, the effect of pH on the experimental rate of hydrogen exchange is assumed to give information about the effect of pH on the equilibrium between A1 and A2.

Harrington et al. 12 have stated that the Linderstr~m- Lang model may be an oversimplification. Emery, 2° in his work on cyclic peptide chelates, has presented evidence which opposes the LinderstrCm-Lang model of hydrogen exchange. Emery doubts the presence of an "opened up" exchanging form of the molecule, but rather believes that the exchange takes place directly from a single conformation. The slow rate of exchange of a given amide hydrogen is a reflection of its local environment. Because of these local environmental factors the pH-dependence of the intrinsic exchange rate of a given amide group may vary from one group to another.

Klotz and co-workers have studied the hydrogen- deuterium exchange properties of N-methylaeeta- mide, 2'-:~ polypeptides, 24 and a polymeric amide, polyisopropylacrylamide35 The major conclusion that Klotz has drawn from his studies is that in addition to hydrogen and hydroxyl ion catalysis of the exchange there is also general acid and base catalysis. I t is suggested that in proteins the acidic and basic side chains create local environments with high concentra- tions of general acid or base and that the state of the solvent in the local environment is not the same as it is in bulk solventY ,25

Two exceptions that have been found to the Linderstr~m-Lang model of hydrogen exchange are myoglobin 26,27 and bovine plasma albumin38,29 These two proteins do not follow the rule 3° that the exchange increases steadily with increasing pH above pH 3. The fact that in myoglobin a minimum in the exchange was found around pH 7 has been attributed to acid denaturation, n Beychok et a lY used optical rotatory dispersion (ORD) to determine that the a-helical content of this protein at low pH is 30~ compared to 75-80% for the native protein. Bovine plasma albumin has been found to show an increase in u with increasing p i t in the pH range 3-57 s,29 This has been attributed to the N-F transformation, a pH-dependent isomeriza- tion reaction39

A similar interpretation can be given to the results of the hydrogen-deuterium exchange studies on BSM reported here. I t has been found that the rate of hydrogen exchange and the extent of exchange both decrease with increasing pH in the pH range 3.7 to

APPLIED SPECTROSCOPY 247

7.2. These exceptional findings may .be a t t r ibuted to a pH-dependent conformational change in the BSM molecule, as was found with myoglobin and bovine plasma albumin. This interpretat ion is in agreement with the ionic s t rength-dependent conformational change found in the light-scattering studies. 3,~° A decrease in pH from 7.2 to 3.7 would decrease the dissociation of the carboxyl groups and lead to a less ordered conformation. Studies using 0 R D are planned to provide fur ther information on the effects of p t I on the solution behavior of BSM.

1. W. Pigman and G. Tettamanti, 4th Int. Conference on Cystic Fibrosis of the Pancreas (Mucoviscidosis), Berne/Grindelwald 1966, Part I I (S. Karger, Basel/New York, 1968), p. 117.

2. A. Gottschalk, Ed., Glycoproteins: Their Composition, Structure and Function (Elsevier, Amsterdam, 1966).

3. F. A. Bettelheim, Y. Hashimoto, and W. Pigman, Biochim. Biophys. Acta 63, 235 (1962).

4. A. Gottschalk, Nature 186, 949 (1960). 5. Y. Hashimoto, S. Tsuiki, K. Nisizawa, and W. Pigman,

Ann. N. Y. Acad. Sci. 106, 233 (1963). 6. B. Anderson, N. Seno, P. Sampson, J. G. Riley, and K.

Meyer, J. Biol. Chem. 239, PC 2716 (1964). 7. K. Tanaka, M. Bertolini, and W. Pigman, Biochem. Biophys.

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(1966). 9. F. A. Bettelheim, Ann. N. Y. Acad. Sci. 106, 247 (1963).

10. F. A. Bettelheim and S. K. Dey, Arch. Biochem. Biophys. 109, 259 (1965).

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Biochem. 35, 599 (1966). 13. S. W. Englander, Poly-c~-Amino Acids: Protein Models .for

Conformational Sludies, G. ]). Fasman, Ed. (Dekker, New York, 1967), p. 339.

14. G. Tettamanti and W. Pigman, Arch. Biochem. Bh)phys. 124, 41 (1968).

15. E. R. Blout, C. de Loz~, and A. Asadourian, J. Am. Chem. Soc. 83, 1895 (1961).

16. A. Hvidt, Compt. Rend. Tray. Lab. Carlsberg 33, 475 (1963). 17. G. Di Sabato and M. Ottesen, Methods Enz. 11,743 (1967). 18. K. Linderstr0m-Lang, Symposium on Protein Structure, A.

Neuberger, Ed. (Methuen, London, 1958), p. 23. 19. W. P. Bryan and S. O. Nielsen, Biochim. Biophys. Acta

42, 552 (1960). 20. T. F. Emery, Biochemistry 6, 3858 (1967). 21. I. M. Klotz and B. H. Frank, Science 138, 830 (1962). 22. I. 5/[. Klotz and B. H. Frank, J. Am. Chem. Soc. 86, 3889

(1964). 23. I. M. Klotz and B. H. Frank, J. Am. Chem. Soc. 87, 2721

(1965). 24. B. H. Leichfiing and L M. Klotz, Biochemistry 5, 4026

(1966). 25. J. S. Scarpa, D. D. Mueller, and I. M. Klotz, J. Am. Chem.

Soe. 89, 6024 (1967). 26. E. S. Bensou, Compt. Read. Tray. Lab. Carlsberg 31, 235

(1959). 27. S. Beyehok, C. de Loz6, and E. R. Blout, J. Mol. Biol. 4,

421 (1962). 28. E. S. Benson, B. E. Hallaway, and R. W. Lumry, J. Biol.

Chem. 239, 122 (1964). 29. J. F. Foster, M. Soganfi, H. A. Petersen, and W. J. Leonard,

Jr., J. Biol. Chem. 240, 2495 (1965). 30. A. Hvidt, Compt. Rend. Tray. Lab. Carlsberg 34, 299 (1(,)64).

INFRARED SPECTRA

The editorial staff of APPLIED SPECTROSCOPY has made arrangements for publication of infra- red spectra which accompany manuscripts in the Coblentz Society's spectral collection. I t is anticipated that this policy will benefit the scientific community by providing for publication of a larger number of spectra than journal space can permit and by offering systematic indexing of these literature spectra in a single collection, l%ferences to the spectra will be provided wilh each article.

The spectra will be reviewed according to the specifications established for the National Standard Reference Data System EAnal. Chem. 38, No. 9, (August 1966)1 and only those spectra which are good enough to pass Class I I I requirements will be published. These are essentially spectra of pure compounds extending at least from 2.5-15 ~ with no more than minor gaps from mulling agents or solvents. Partial spectra for discussion of a narrow region of the spectrum will continue to be published in the journal article.

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248 Volume 23, Number 3, 1969