Oct., 1959
SORPTION OF H 2 0 AND D20 BY LYOPHILIZED LYSOZYME
1653
THE SORPTION OF H20 AND D20 BY LYOPHILIZED LYSOZYME1 BY WASYLS. H N O J E W YAND J ~ LLOYD H. REYERSON The School of Chemistry, University of Minnesota, Minneapolis 14, Minnesota Received MaTch 6,1069
Isotherm data for the sorption of HzO and D20 on lyophilized lysozyme are reported. The heats of sorption are calculated. Not only is D10 more sorbed than HzO, but the differential heats of sorption show interesting differences.
Earlier work from this Laboratory3 presented data for the sorption of HzO by lyophilized ribonuclease. Calculated heats of sorption gave rather large values for the first percentages of water taken up but these values fell rapidly until about 5.5% of water had been sorbed. Evidence indicated that the break in the heat curve occurred roughly a t monolayer coverage, which meant, as suggested by P a ~ l i n gthat , ~ the polar side groups on the protein had been saturated by water molecules It was felt desirable that this type of work be extended to other lyophilized proteins and that isotherms for HzO be compared with those of D20. This work presents results obtained for the sorption of HzO and D2O by lyophilized lysozyme at 17 and 27".
Experimental
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Sorption isotherms were measured gravimetrically using a quartz spring helix having a sensitivity of 12.580 mg./cm. Displacements were measured with a traveling microscope to &0.003 mm. 169.8 mg. of the protein was used for the HzO sorptions and 176.7 mg. for the DzO study. The lysozyme was obtained from the Armour Research Division of the Armour Company (Lot D-638040) and was lyophilized from about a 3% solution in triply distilled, deionized water at a pH of 6.8 t o 7. It was frozen dried on a fine glass rod using an alcohol-COz mixture. The glass rod containing the protein was attached t o the hook at the lower end of the quartz spring balance. After careful outgassing at a pressure of 10-6 mm., the isotherms were determined in the same manner as previously described,a except that in this case the measurements were carried out at 17 and 27'. The purified .HzO and D,O (99.5%) were outgassed by a series of freezings, thawings and evacuations.
Results and Discussion Since comparisons between HzO and DzO were being made, the isotherms for the sorptions of both vapors are given in Figs. 1 and 2. I n the case of the isotherm of HzO a t 27", the vapor was sorbed up to 18.7y0 of the weight of the dry protein. The desorption that followed was slow but it was found possible to finally return to the original zero point in spite of the marked hysteresis, as shown in Fig. 1. At 17" the sorption was carried to a much higher vapor pressure and, as a result, to a much higher uptake by the protein. Even a t an adsorption of 58% of the protein's weight of HzO,the shape of the protein on the glass rod remained essentially unchanged except that a slight swelling was observed. The ribonuclease used in the previous study formed a solution in the water when it had taken up about this amount of the vapor. I n the case of the lysozyme, when the uptake of H 2 0 reached about 70y0, the fluffy white protcin on the rod collapsed. The desorption hysteresis shows a difference from that a t (1) This work was supported by a Research Grant from the National Institutes of Health. (2) . . Research Assooiate. Universitv of Minnesota. (3) J. G. FOES and L. H. Reyersoh, ~111sJOURNAL, 62,1214(1958). (4) L. Pauling, J . Am. Ckem. Soc., 66,555 (1945).
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27" but it was found possible to finally reach the same zero point in spite of the longer time needed.
WASYLS. HNOJEWYJ AND LLOYD H. REYERSON
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Heats of adsorption of water-" and deuterium
The collapse of the protein certainly altered the There is an initial maximum for each vapor and porous structure of the protein and there could have this is nearly twice as great for HzO on the lysozyme been some internal changes, but the desorption iso- as was found for H2O on ribonuclease. The maxitherm does not show too significant a difference. mum for the H20 calculates to be about 30 kcal. It was apparent, almost immediately after begin- per mole, while for D2O the value turns out to be ning the studies using D20, that the protein sorbed about 24 kcal. The heat values fall for both as more DzO at the same vapor pressure than it did more vapor is adsorbed, but the rate of fall is much H2O. The second sample may have had a differ- greater for HzO and the two heat curves cross a t an ent surface, even though the two were prepared uptake of 5% where the heat value is 19 kcal. Bealike. The first isotherm a t 27" carried the increase yond this point the differential heat is higher for in weight to about 7.0% of the weight of the pro- DzO than for H2O until the two curves meet a t tein. Upon desorption, under the same conditions between 13 and 14% uptake. Here the heat value as for HzO,it was found that the weight of the pro- is 12.5 kcal. There are no values beyond this tein did not return to the same zero point. I n this point for DzO, but those for HzO continue to fall first case, the permanent addition of weight slowly until 19% uptake where the heat is close to amounted to 0.587% of the initial weight. A sec- that of the heat of vaporization of H2O. Studies ond sorption then was determined, with the amount are being continued over a wider temperature range of added D20 reaching approximately 14% of the in the hope that a reasonable explanation may be weight of the protein. Upon desorption, the pro- arrived a t for the differences between the sorption tein retained DzO equal to 1.10% of its original of DzO and HzO. weight. The final isotherm a t 17" was carried up In conclusion, one may say that HzO is more to about 18% of added D20. Upon desorption, the strongly sorbed by lysozyme at the low vapor presprotein retained D2O equal to 1.37% of its initial sures than is DzO. The differential heat calculations weight. Since the protein sample was the same for show that in the initial uptake of vapor the heat of all three isotherms, this meant that an increase in sorption of H20is greater than that of D2O. Howweight occurred during each adsorption-desorption ever, the heat curves cross at 5% uptake and that cycle. This strongly suggests that deuterium ex- of D20 remains above that of H20until the values change took place on the surface of the lyophilized approach the heat of vaporization of H2O. A reaprotein, between certain of the hydrogen atoms of sonable explanation of these results is being sought the protein and the deuterium of the adsorbed D2O. by additional studies. Using the data from the isotherms for H 2 0 and DISCUSSION D20 at 17 and 27", the differential heats of sorption were calculated and the values plotted, as W. HELLER(Wayne State University).-How long did it shown in Fig. 3. The dashed sections of the curves take t o reach equilibrium? were used to indicate that the precision attained in L. H. Reyerson.-From 4 t o 6 hours and in some cases as the initial adsorption values a t low vapor pressures much as a day was required. was not quite good enough to make the heat value W. HELLER.-HOW thin was the layer of protein? calculations exact. The solid lines in Fig. 3 are considered to be reasonably good. The heats for L. H. REYERSON.-It was a rather loose, cotton-like the initial uptake of H2O are higher than for D2O. porous maRs hanging on a glass rod.
