老妈花了4K买了个电脑包...还打算拿4K买个电脑.我立刻驳她:你敢把4K的电脑从你4K的包包里拿出来么?其实你只是想要那个包而已,你甚至可以不买电脑...老妈恍然大悟.决定去买1W的电脑和4K的包...

又一夜没睡,支撑到现在,早上八点去找了导师,说了情况,没想到老人家这么好说话,成功请到假.回宿舍洗个澡,继续赶校车回家.车站有二十多号女子,就我一男的,一种异样的恐惧感油然而生,排队上车的时候,某女子说,就你一个男的,你先上吧...接着就是一片放肆的笑声.就突然感觉自己好弱势...阿门...

下午导师助理打来电话，要求七月一日就去实验室。霎时间，情绪降至冰点。老爸晚上也送来了机票，说了句，不能退的啊。我居然直接上床就睡觉了，睡到一点钟起，一边整理电脑，一边想今天Naric本周来第二次的警戒：你马上就要没有市场了，一边想Parry淡定的通知我她恋爱了，一边想今天好像忘记给梅梅打电话，最后在想白天怎么用苦肉计去给导师求情放我回家去。然后扫了一眼表发现已经五点了...

DT那点破事，你丫的我早就知道了。不管发生什么，后果都要自己承担，我们总是片面的，不客观的在处理你遗留下来的事物。

好歹今天也是你的生日，破手机又开始当板砖了啊，只好在这里祝你生日快乐了。下不为例。

首先解释一下上一篇日志。一篇遭人轮的日志。。。

毕业设计要求一篇外文论文的翻译，由于本人不慎选择了一篇过长的文章，plus，写于1944年的古董。逐字逐句的翻译又有些紧迫，于是我贴在了博客上，让google翻译我的网页。然后ctrl c +ctrl v到我的文档中再修改。本人并无卖弄之意。却忘记即时删除，结果迎来太多怨念，我也只好保留以示警戒。翻页的结果让人更加xx，五花八门，错从复杂。着实让我自愚了一把。

总之，一切结束了，研究生录取结束，论文结束，答辩结束，聚餐结束。唯一要做的，就是开始安排度日的行程。

naric搬了新家，姐姐也要结婚了，parry的offer也在路上了，买买依旧沉溺在爱情绿洲中，titi姐被街拍了(这可是相当的即将要必然要轰动的)，downtown和我玩了两会儿wow再也不见踪影了。之前太忙，几乎忘记了联络很多朋友，现在突然闲下了，罪恶感出现了，碰巧今天在正午时分33度的马路边接了一通电话后，我的汗液渗入了手机，让我的被许多人称为小8而我称为38的N81崩溃了。真是可以呢。真的。上网搜索后发现除了无数人有同样的情况而没有解决方法，就是无数喷子对诺基鸭子的客服的口水。于是我放弃了，先晾一个晚上再说。

听说越狱结束了，史高德终于可以不被我身边的人群们提及了。我爱上了无脑的聚集，CSI类的和The Big Bang类的，虽然类型不同，但是共同点就是，我不用记住上一集讲了什么，你可以从任一季的任一集开始开起，而且每集只有一个主题，不给你猜测剧情的权利，不让你淹没在未知的思绪翻滚中。

so，i am fine with all of this.

ISOLATION OF LYSOZYME FROM EGG WHITE

BY GORDON ALDERTON, W. H. WARD, AND H. L. FEVOLD

(From the Western Regional Research Laboratory,* Albany, California)

(Received for publication, October 2,1944)

In 1922 Fleming (1) applied the term lysozyme to a bacteriolytic agent found in the tissues of a number of species of animals. The highest concentration of the agent has been found in hen’s egg white which, in a dilution of about 1:500,000, has the power to dissolve a thick suspension of test organism.

Previous chemical work has been concerned only with the active principle from egg white. Available evidence, contributed mainly by Abraham (2),indicates that lysozyme is a basic protein with a molecular weight near 18,000. Abraham reported that it is stable when heated in acid solution but very heat-labile in alkali. Acetone, ether, and alcohol precipitate the active material from aqueous solution without destroying it. This latter property is the basis of the methods of preparation devised by Roberts (3) and Meyer et al. (4). In these methods the initial purification is obtained by rendering most of the egg white proteins insoluble by selective acetone denaturation at the alkaline reaction of native egg white. In the method of Meyer et al. (4) the precipitated proteins are first extracted with a mixture of alcohol and aqueous acetic acid. This step is followed by concentration of the extract under a vacuum, precipitation with alcohol, solution of the precipitate, and isoelectric precipitation of inactive protein with sulfuric acid. The lysozyme is then precipitated by flavianic acid. Repeated extraction of the dye-lysozyme complex with alcoholic ammonia liberates the active portion of the complex. The insoluble residue containing lysozyme is freed from ammonia by washing with alcohol and ether, followed by drying under a vacuum.

Investigations dealing with the therapeutic usefulness of lysozyme have been reported by Russian workers (5). They report favorable results from treatment of ulcers of the eye, postoperative infections, burns, infectedwounds, and sinus infections. It appears, therefore, that lysozyme mightfind wide application if sufficient material could be made available inpurified form. The present communication reports the results of work which was initiated for the purpose of developing a method for the preparation of large quantities of lysozyme. The method reported here makes possible the recovery of lysozyme from egg white in high yield, and the * Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, United States Department of Agriculture.product appears to be essentially pure. In addition, the product has been obtained in apparently pure crystalline form.

EXPERIMENTAL

Purification of Lysozyme by Salt Fractionation-Attempts were made to purify lysozyme by fractionating the egg white with ammonium sulfate, but with indifferent success. The characteristic of lysozyme that militated against salt fractionation was its adsorption in varying degrees on inactive protein precipitates. The effect of pH on the ammonium sulfate precipitation was investigated but failed to reveal conditions that would prevent adsorption of lysozyme. Likewise, dialysis against buffers of different strength and hydrogen ion concentration did not prevent adsorption.By repeated solution and reprecipitation of euglobulin fractions containing adsorbed lysozyme, preparations with an activity 25 to 30 times that of egg white, on the dry weight basis, were obtained in low yield.1

Purification of Lysozyme by Adsorption and Elution Methods-Fleming (1) reported that lysozyme is readily adsorbed on charcoal, cellulose, and porcelain, and subsequent studies have shown that lysozyme is adsorbed tenaciously on many substances. Wolff (7) found that norit, kaolin, silicic acid, and filter paper adsorb the active material. He was unable, however, to effect elution from kaolin or charcoal, irrespective of pH. In studies on the purification of lysozyme, the Russian investigators, Buyanovskaya and Jermol’eva (5), attempted to make use of the ready adsorbability of lysozyme on clay. At pH values ranging from 5.5 to 9.0,

lysozyme was adsorbed on kaolin suspended in buffer solutions, including phosphates, glycocholates, acetates, and dilute ammonia solutions. Treatment of the kaolin-lysozyme complex with various buffer solutions ranging from pH 4.5 to 14 did not elute the active material. Attempts to separate the positively charged lysozyme from the negatively charged clay particles by cataphoresis were unsuccessful.

We investigated the capacity of several adsorbing materials to bind lysozyme. Among these were bentonite (a very finely divided mont-

1 While this preparation was not investigated further, comparison of its activity with that of crystalline lysozyme obtained later showed that it was of high purity.The method of Boasson (6) was used for assay purposes. By this method the rate of lysis of a suspension of killed Micrococcus Zysodeikticus organisms is measured as indicated by the rate of change of light transmission recorded by a photoelectric calorimeter. Under standardized conditions a given change in galvanometer reading

over a certain time interval corresponds to a definite amount of lytic agent. A lysozyme unit is defined as the amount of lysozyme in 1 ml. of a standard sample of liquid egg white. However, the amount of lysozyme in fresh egg white has been

found to be very constant; so the unit might be stated to be the amount present in 1 ml. of fresh egg white. As routinely carried out in our work the method was reliable to approximately 15 per cent.

morillonite clay), Supersorb (a synthetic zeolite), Panther Creek bentonite,and Duolite (a synthetic resinous cation exchanger). Of the substances investigated, bentonite was found to be the most efficient adsorbent for lysozyme from native egg white. 1 gm. of the material removed the active substance from 80 to 100 cc. of egg white. Table I gives the data from a number of representative experiments.

We have confirmed the results of previous workers in that we were unable to elute the lysozyme with aqueous inorganic buffer solutions. Potassium chloride solution and 6 M urea solution likewise did not removeany active material. Since organic bases are known to be strongly adsorbed by montmorillonite clays, solutions of o-phenylenediamine and p-phenylenediamine were tried but the amines were adsorbed on the clay without eluting lysozyme. However, a mixture of 20 per cent pyridine in 2 per cent aqueous acetic acid did elute about 30 per cent of the adsorbed activity.The pH of this solution, as determined by a glass electrode, was 6.0.

Investigation of the effect of pH on the elution of lysozyme by pyridine solutions proved that it could be eluted very efficiently at the proper pH. In these experiments acetic acid was replaced by sulfuric acid in order to extend the pH range. Rliquots of a suspension of clay on which lysozyme had been adsorbed were stirred with pyridine-sulfuric acid solutions at various pH values. The eluates were removed from the clay and assayed for lytic activity.

The elution of lysozyme was found to be specific with regard to the pH of the eluting solution (Fig. 1). When the pH is lowered, removal of the active material begins at pH 6.2, rises sharply to a maximum at pH 5.0, and falls off to zero at pH 3.0. Appreciable amounts of inactive protein are eluted by pyridine or pyridine-sulfuric acid solutions at pH values above 6.2, thereby effecting a marked purification of the adsorbed bactericide.

Based on the facts reported above, the following method was devised for separating lysozyme from other egg white proteins. To each liter of egg white are added 150 ml. of a 10 per cent suspension of bentonite in 1 per cent KC1 and the mixture is stirred vigorously (foaming being avoided) for 3 to 5 minutes. The clay is separated from the suspension in a Sharples centrifuge and washed with 0.5 M phosphate buffer at pH 7.5 to remove egg white mechanically held in the clay mass. The clay is next washed three times with 300 ml. (total 900 ml.) of a 5 per cent aqueous solution of pyridine to remove inactive adsorbed proteins. Elution of lysozyme is then accomplished by washing the clay twice, each time with 300 ml. of a 5 per cent aqueous solution of pyridine, which has been adjusted to pH 5.0 (glass electrode) by the addition of sulfuric acid. Removal of the eluates from the clay is accomplished by centrifuging in a batch type centrifuge.For best results elution should follow adsorption within 24 hours.

FIG. 1. Effect of pH on elution of lysozyme from bentonite

Typical data on distribution of protein solids, yield of lysozyme, and activity of the preparations obtained are given in Table II. These data show that as high as 90 per cent of the lysozyme present in egg white can be obtained by this process in a concentration of 35 to 40 times that of the original egg white solids. The activity of the material is constant from one preparation to another within the limits of error of the assay method.

Concentration of the active material contained in the eluates at pH 5.0 may be accomplished in a number of ways. Addition of ammoniumsulfate to a concentration of 2.6 M precipitates the active substance, which may be collected by centrifugation or filtration. The precipitate readily dissolves in distilled water and the solution may be freed from salt by dialysis . Drying the solution in the frozen state gives a white solid, of the activity indicated, which remains stable at room temperatures for several months. Concentration can be effected also by precipitation with 75 percent alcohol or acetone, preferably in the cold, followed by dialysis.

We have found it more convenient to prepare the dry lysozyme powder in the following manner. The active eluates are placed in cellophane bags and dialyzed against running tap water until no odor of pyridine remains. The dialysis is then completed against running distilled water for 24 hours. The solute, after being dried from the frozen state, is a light, fluffy, stable powder.

Fractionation of Purified Lysozyme with Ammonium Sulfate-h an attempt to purify the active material further, two 1 per cent solutions of the eluted lysozyme in distilled water, adjusted to pII 5 and 7, were fractionated with ammonium sulfate. The salt was added by dialysis through a rotating membrane (8) at I”, and precipitates were removed at increments of 0.2 M ammonium sulfate concentration and the solids and activity determined on the precipitates and filtrates (Table III). No significant concentration of activity was observed in any of the fractions, the activity of each being essentially that of the starting material within the limits of the determ.inations. The least and t,he most soluble fractions appear to have less activity than the rest of the material but the amounts of solids in these fractions were so small that the accuracy of the analysis is questionable. It appears from these experiments that the preparations obtained on elution were grossly homogeneous.

* Egg white contains 0.1 to 0.12 gm. of protein solids per ml. and 8 to 10 lysozymeunits per gm. of protein.

Action of Enzymes on Lysoxyme Preparation-It has been reported that lysozyme is not destroyed by proteolytic enzymes (5). Since it is non-dialyzable, it should be possible to remove digestible contaminating proteins by enzyme degradation followed by dialysis. Accordingly, 10 ml. portions of a lysozyme solution containing 8 units per ml. were digested for 24 hours at 30” with bacterial proteinase (pH 7.4), mold proteinase (pH 7.4), trypsin (pH 7.4), papain activated with cysteine (pH S.O), and pepsin (pH 2.0). After the activity of each digest had been determined, each was dialyzed

against distilled water for 16 hours and the solids determined. The activity per unit of undigestible material was then calculated.*2

No significant loss of activity took place on digestion with any of the enzymes, with the exception of pepsin. Similarly no marked loss of material took place on dialysis after digestion except in the case of the pepsin-treated material. The calculated activity of the undigested material was in all cases not significantly different from the determined activity before digestion (Table IV).

Two conclusions may be drawn from these experiments. Of the enzymes used, only pepsin digests lysozyme at a rate measurable under the conditions of the experiment, and no proteins that are digested by the enzymes used, other than pepsin, were present in measurable quantities. Only small amounts of inactive, pepsin-digestible protein might be regarded as

2 Unfortunately the activity after dialysis was not determined, but since lysozyme does not pass through the membranes it appears permissible to ascribe the activity measured before dialysis to the undialyzable fraction.

having been present, since no increase in activity of the undigested material was apparent. This conclusion is valid, however, only if the rate of digestion of the inactive material is greater than that for lysozyme.

Since many proteins are sensitized to enzyme action by heat, experiments were carried out to determine whether the protein or proteins in the lysozyme preparation could be thus sensitized and a differential digestion then be obtained.

*Heated in HCl(pH 3.0) at 96°

Lysozyme has been reported to be stable to heat in acid solution but very labile at alkaline reactions. Before proceeding with the digestion experiments, we determined the effect of heating alone on the activity of lysozyme. Lysozyme powder at pH 2.8 was dissolved in distilled water and heated on a steam bath at a temperature of 96”, and samples were removed for analysis at regular intervals. While the rate of destruction was slow, a progressive decrease in activity was found which amounted to a 60 per cent loss after 80 minutes of heating (Table V).

A lysozyme solution similar to that used for the preceding experiment was heated until a destruction of 40 per cent had taken place. The solution was then treated with the various enzymes in the same manner as described for the unheated material. The results are presented in Table VI. Apparently the rate of digestion by all the enzymes had been markedly accelerated. After removal of the digested part by dialysis, however, the activity of the undigested remainder was the same as that of the original unheated material with one exception; that is, when papain was used. In this case the unit activity was reduced, which may mean that digestion of the material, inactivated by heat, was not complete or the products of digestion were not completely dialyzable. In no case was there any indication of a differential digestion resulting in increased activity of the undigested active preparation. It is also evident from these experiments that some change in the lysozyme molecule is induced by heating which does not disrupt the structure necessary for lytic activity but which does sensitize the molecule to enzyme action.

* Heated in HCl (pH 3) at 98° for 90° minutes.

t Calculated as per cent of activity after heating.

Electrodialysis of Lysozyme Solutions-Solutions of lysozyme which had been dialyzed against distilled water were further freed from electrolytes by electrodialysis. The electrodialysis was carried out at 1’ for approximately 18 hours, or until no appreciable change in conductivity of the solution took place. The pH, which was approximately 7.0 at the beginning, had risen to approximately 9.5 at the end of the experiment, and some precipitation of the solute took place. With adjustment of the electrodialyzed solution to pH 10 to 10.5, heavy precipitation resulted. At pH 11 resolution of the precipitate was apparent, and was complete at approximately pH 11.5. At pH 10.8, which appeared to be the point of minimum solubility, 6 mg. per ml. remained insolution, whereas, at pH 9 and 11.5, 150 mg. per ml. readily dissolved. No difference between the lytic activity of the precipitates and that of the material remaining in solution in the isoelectric region could be demonstrated. Therefore, no evidence for the presence of more than one component was found.

Longsworth, Cannan, and MacInnes (9) have reported that electrophoretic analysis of egg white proteins reveals the presence of one and only one component with an isoelectric point above pH 7.0. This component was designated by them as G1. The average amount of this component comprised 2.8 per cent of the egg white proteins, whereas the average yield obtained in our preparations was 2.5 per cent of the proteins present in egg white. While the mobility of G1 was not determined by Longsworth et al. above pH 7.8, extrapolation of the mobility curve shows an isoelectric region around pH 10.5 to 11.0, which is in close agreement with the isoelectric point of the prot,ein contained in our lysozyme preparations. These facts point strongly to the identity of G1 and lysozyme.

Electrophoretic Behavior and Isoelectric Point of Lysozyme-Several purified lysozyme preparations have been analyzed electrophoretically by the Tiselius met,hod within the pH range of 4.5 to 11.8 and at an ionic strength of 0.1. In all of these analyses one component comprising more than 95 per cent of the total gradient was present and in several the mat.erial appeared quite homogeneous. In most of the analyses the ascending boundary appeared homogeneous, while in one or two trace components could be detected. The descending boundary in many of the analyses spreads considerably, beginning abruptly at the leading edge, sometimes with a small spike. It was in these spreading boundaries that evidence of the presence of trace components was chiefly observed (see foot-note to Table VII and Fig. 2). The conclusion seems justified that the lysozyme preparations are essentially electrophoretically homogeneous and may consist of quite pure protein. The results of all analyses are given in Table VII.

The mobilities of lysozyme approximate the values reported for the globulin component of egg white designated G1 by Longsworth, Cannan, and MacInnes (9), as shown in Fig. 3. In the acid buffers the mobilities were lower than reported for G1, but in Analyses 1 and 2 of Table VII the sharp leading edge coincides with this reported mobility. Phosphates depress the mobilities of lysozyme and G1 from the values in buffers containing only monovalent ions to approximately the same extent. In the media near pH 7.8, two different samples were found to have mobilities closely similar to that reported for G1, although differing in mobility from the lysozyme preparation reported by D. Moore in an article by Meyer (+6.75 X 10-j sq. cm. per volt second at pH 7.80) (10).

The properties of G1 at higher pH were not discussed by Longsworth and his associates, except for the statement that an isoelectric point was not observed. We found that lysozyme moved toward the anode in a medium of 0.01 N NaOH and 0.09 N NaCl (pH 11.80) in which its biological activity

* Acids and salts present in buffers used were as follows: A = acetate, C = dimethylarsonate

(cacodylate), V = diethylbarbiturate (barbital, veronal), E =ethanolamine, G = glycine.

t Mobilities are reported in terms of 10-G sq. cm. per volt second, referred to 0’

by multiplying the mobility at the temperature of observation by the viscosity of

water at that temperature relative to.the viscosity of water at 0”.

$ In several analyses the falling boundary spreads considerably, beginning abruptly

at the leading edge, sometimes with a small spike. The rising boundary was

sharp in these cases, so that the spreading may be attributed to imperfect ionic

adjustment of solution and buffer, and the spike may not represent an extra

component.

$ The sample, isoelectrically precipitated, was apparently quite homogeneous in

this medium, but in Analysis 6 showed trace components, as noted.

11 Mobility of minor component estimated from rising side.

7 Nearly saturated solution.

** Apparently homogeneous, but the total displacement during the time available

for analysis was insufficient to allow as conclusive a test as in other instances.

it Analysis of the solution for biological activity and nitrogen content indicated

that the lysozyme was stable under the conditions of the experiment.

FIG. 3. The electrophoretic mobility of lysozyme as a function of pH, compared with that of the globulin G1.

remained constant. Extension of the published graph to the mobility value at this pH indicates an isoelectric point near pH 10.5. However, the mobility of lysozyme in buffers containing ammonia, glycine, or ethanolamine, between pH 9.5 and 11, is regularly toward the cathode and faster than indicated by this extension of the trend of values at lower pH. The isoelectric point in these media seems to be near pH 11.0. This circumstance suggests complex formation with the amino constituent of the medium, just as the deviation with phosphates suggests phosphate binding.

The correspondence of the electrophoretic and chemical properties of the highly purified lysozyme with those of the globulin G1 indicates that they are very similar and in fact may be identical.

Sedimentation, Diffusion, and Osmotic Pressure Measurements with Lysoxyme-Solutions of the purified lysozyme were studied in an air-driven ultracentrifuge, a Lamm-Polson diffusion cell (ll), and in a modified Hepposmometer (12, 13).3 The measurements indicated that the lysozyme preparation is homogeneous and that lysozyme falls among the lowest weight proteins listed by Svedberg (14).

From the sedimentation rates measured at lysozyme concentrations of from 1.5 to 0.5 per cent, in 0.15 M sodium chloride, values at A!!&, W were found to be about 1.9 Svedberg units. The average diffusion constant, 020, W, calculated from the maximum scale line displacement and area, and by the method of successive analysis (( 11) Equation 50) is 11.2 X lo-’ at 1 per cent lysozyme concentration. Partial specific volume measurements have not been made, but if we assume values from 0.70 to 0.75 we can calculate molecular weights from 14,000 to 17,000 from the sedimentation and diffusion constants obtained for this material.

The osmotic pressures were determined by extrapolating to zero time measurements with cellcphane membranes (No. 300)) through which lysozyme diffuses slowly. The pressures obtained were extrapolated to zero concentration of lysozyme by a plot of the ratio of pressure to concentration versus concentration. The measurements made at two concentrations of lysozyme gave the value of the ratio of pressure to concentration equal to 151. From this value a molecular weight of 17,500 is obtained.

Since partial specific volumes were not determined and osmotic pressure measurements were made with a’slightly permeable membrane, the values for the molecular weight of lysozyme must be considered as approximate. However, they are in agreement with each other and also with the molecular weight of 18,000 given by Abraham (2).

Crystallization of Lysozyme-Lysozyme has been obtained in crystalline form. Crystallization has been successful under a wide range of pH values, ranging from the isoelectric region (pH 10.8) to 3.5. Two factors contribute to the ease of crystallization of lysozyme. It has a relatively high positive temperature coefficient of solubility, and the solubility of the amorphous material is 3 or 4 times as high as that of the crystalline form.

3 Davis, B. D., personal communication.

Crystallization at pH 4.5 was carried out in the following manner. 4 gm. of isoelectric protein, which had been dissolved in dilute acetic acid at pH 6 and dried in the frozen state, were dissolved in 60 CC. of 0.2 M acetate buffer at pH 4.5, containing 5 per cent of sodium chloride. The solution was allowed to stand at room temperature, when well defined crystals were deposited as shown in Fig. 4. Only 15 mg. per ml. of the original material remained in solution after crystallization for 16 hours. The rate of crystallization may be markedly increased by lowering the temperature from 21° to 4°.

Crystallization at the isoelectric region (pH 10 to 11) was accomplished by agitating an excess of the isoelectric protein with saturated sodium chloride adjusted to pH 11 with XaOH. The amorphous material partially dissolved, and as a result of the lower solubility of the crystalline

FIG. 4. Crystalline lysozyme (120 X). Crystallized from 0.2 M acetate buffer,pH 4.5, containing 5 per cent NaCl.

product, crystals were deposited. When the process was continued long enough, all of the excess amorphous solid was changed to the crystalline state. The crystals so formed were very thin rectangular plates.

Crystallization from ammonium sulfate solutions (0.4 M) buffered at pH 5.0 with acetate, or at pH 7.0 with phosphate, resulted when the temperature was lowered from 21” to 4”. Crystallization was also effected at room temperature from 1.4 M ammonium sulfate solutions acidified to pH 3.5 with sulfuric acid. The activities of the various crystalline preparations

were in all cases similar to those of the amorphous material.

The crystalline forms obtained by the procedures outlined above seem to vary with the pH at which crystallization is carried out and with the acid used in effecting solution of the isoelectric material. Since lysozyme is a very basic protein, it seems that the crystal form of its salts with various acids may vary. This matter is being further investigated and the results will be reported later.

DISCUSSION

It is difficult if not impossible to compare the activity of our preparations with those reported by other investigators. Meyer et al. (4) stated that their preparations contain from 2 to 6000 units per mg. The unit is defined as the smallest amount causing complete lysis of Micrococcus lysodeilcticus in a serial dilution test. The quantitative yield of active material is not given. Abraham (2) reported Robert’s preparation (3) to be an improvement over that of Meyer et al. (4) and to behave as a homogeneous protein in the ultracentrifuge. Robert’s unit is similar to that of Meyer et al., and the activities are likewise incapable of translation, since percentage yield and activity of egg white are not given. Also, in our preliminary work with assay methods we found that the susceptibility of Micrococcus lysodeikticus varied many- fold from day to day and from culture to culture, and hence found it necessary to use dead organisms in order to carry out quantitative work.

Abraham and Robinson reported in a short note in 1937 (15) that crystalline material was obtained from a lysozyme preparation which was prepared by Robert’s method (3). This preparation appeared to be homogeneous in the ultracentrifuge, but Abraham (2) was able to separate two fractions of different solubilities and activities. Crystallization was induced by evaporation of a 0.05 N acetic acid solution in vacua over aqueous KOH. 2 years later, however, Abraham (2) reported that he had not been able to obtain sufficient crystalline material for chemical examination. The fact that we have been able to obtain no evidence for the presence of two substances of varying activities and the ease of crystallization of our purified materials indica,te that the two preparations may

not be identical.

The facts presented in this paper indicate that lysozyme and the globulin called G1 by Longsworth et al. are identical. The amounts present, the isoelectric region, and the mobilities are all in approximate agreement. It appears unlikely from available information that more than one substance with these characteristics is present in egg white in the amounts found for G1 and lysozyme.

While this paper was in preparation, two papers (10,16) appeared dealing with the relationship of biotin, avidin, and lysozyme. In the preparations used in the work reported in these papers it seems that avidin activity and lytic activity were correlated, and it was intimated that lytic activity may depend on the presence of avidin and biotin. In preliminary experiments with the pure lysozyme preparations we have been unable to obtain any indication that avidin and biotin are concerned in the lytic activity of lysoeyme. These experiments will be reported more fully in a later communication.

SUMMARY

1. A method for the isolation of lysozyme from egg white, in high yield and in essentially pure form, has been developed. The method depends on the (a) adsorption of Iysozyme on bentonite (a montmorillonite clay), (6) elution of inactive contaminating proteins from the clay by successive washings with phosphate buffer (pH 7 to 8) and 5 per cent aqueous pyridine, and (c) elution of the active material with pyridine-sulfuric acid solution at pH 5.0. The eluate is dialyzed and dried in the frozen state. A white powder is obtained containing 85 to 90 per cent of the lysozyme contained in the egg white.

2. The lysozyme preparations have been shown to be essentially pure by salt fractionation, by their behavior toward enzymes, and by electrophoretic and sedimentation studies.

3. Lysozyme is a basic protein of low molecular weight (about 17,000). It is isoelectric at some point between pH 10.5 and 11.

4. The purified substance is stable in acidified solutions and relatively stable also in alkaline solutions. At pH 11.5 no loss of activity could be detected over a period of 5 to 6 hours.

5. Lysozyme has been prepared in crystalline form. Crystallization has been effected at the isoelectric region (pH 10.8), at pH 7.0, and in acid solutions (pH 3.5 to 5.0). The crystal form appears to vary, depending on the pH of crystallization and the acid used in dissolving the protein.

6. It seems probable that lysozyme and the substance termed G1 by Longsworth et al. are identical.

We are very grateful to Dr. J. L. Oncley, Department of Physical Chemistry, Harvard Medical School, Boston, Massachusetts, and Dr. A. Brown, Laboratory of Physical Chemistry, Massachusetts Institute of Technology,Cambridge, Massachusetts, for carrying out the sedimentation, diffusion, and osmotic pressure measurements reported in this communication. We are also indebted to E. Wolford and F. T. Jones of the Western Regional Research Laboratory for the preparation of the Micrococcus lysodeikticus organisms for test purposes and the preparation of the photomicrograph Respectively

一直以来水果都让我提不起兴趣，有时候即便是剥好的也不想碰。当然这是有严重代价的，大便书教育我们这样的便便是不会健康的。

于是前天我走进超市在完全不自主的情况下奔向了卖水果的地方，买了芒果和菠萝，回去即时消灭掉。这就是动物的本性，我猜。看来我是缺乏维生素了。

当问题涉及到目前的工作进展，我已经觉得有点度日如年了，每天朝九晚五只是为了过去在实验室里发呆，我现在已经严重厌倦了这样的生活，无奈我还有两个月要呆在那个没有信号的地方，饱受被奴役以及食堂的折磨。眼看试验进展到最后一步，Lucas却病的惨不忍睹，无奈试验延期进行。

哦，天哪。我隐约觉着现在上网也是个负担，思考上网干什么也是在考验我的想象力。昨天晚上更是奋笔三个小时去做一件丝毫不感兴趣的事情，我突然很想吃东西，用饱这个状态填补我精神上的无聊。听起来还不错，不是么。

Parry的手机终于灵异了起来，Mary的间歇性心理低潮期也终于爆发了起来，不过这一切都会过去的不是么？我在问谁呢...这一切都会过去的，用这句俗气的真理来概述一切问题虽然显得有点不进取但是这的确是你毫无头绪面对困难时的救命稻草。我扯远了。

写了三周的开题，修修改改，终于开始做试验了，还被同学鄙视：从鸡蛋清里提蛋白？听起来好低级哦。早上来实验室打了两个鸡蛋清准备今天的试验，看着黄油油的鸡蛋黄觉得很可口，立马有念头去超市买瓶VODKA装逼做鸡蛋酒，突然感觉自己像是Karen Walker一般离不开酒。我不禁联想自己以后是一个酒鬼般的科学装逼贩子。

午饭食堂再次毫无悬念的给我们打雷，胡萝卜萝卜丝这种菜名出现在小黑板上，小沈阳和甘大叔在一边立刻爆出萝卜开汇的耸词。超市里又有新发现，Kinder这个巧克力，包装上一白牙男童一副好满足的样子，吃了以后我发现这玩意儿真的挺歹的。看看包装，丫的还是德国来的。说道巧克力，去年情人节在家买的那比利时的，可惜再没发现过，那天在学校附近的超市发现了中包装的，七十多块，本来想买，想着超市暴利就犹豫了，想上网看看，结果发现网上卖更贵。超市现在好洋气，充斥着进口食品，但大部分是一堆高丽棒子的，直接无视。可是最关键的是有干白卖。

每天总是看到半夜的美剧，白天打发时间。在荒废了新的一天后，我总能安然入睡，心中默念这样挺好的，想想这是在半年之前根本不可能的事情，周围的人们都在忙着自己的事情，我就恰巧陷入了尴尬的空白阶段。老妈说活该，但又每天一通电话生怕我想不开。我也觉得活该，为什么追逐自己不想要的事情，而且又那么不专业的追逐着。难道我要转型，在看到了实验室人生的悲惨世界后，我又跳出了压抑很久的念头，只是貌似现在没有煽风点火之势，这念头也就奄奄一息了。to cross or not,that's bothering，但我又很满足现状，并且觉得自己可以把自己磨平了适应，只要给我钱，只要给我足够的空间，我想我是可以很好的活下去。懒得想了，废话时间。

乳娃娃的生活：

早上七点起床，把自己从昨天下班到睡醒阶段的狗模人样的状态变成即将奔赴单位的人模狗样的状态。

七点半下楼吃早饭，食堂推出新品：烤鸭卷，估计我可以用一个月时间吃厌它。

八点准时做校车去正门口公交车站，花一个小时车程颠簸到上班的地方。

九点到实验室，看今天的手机报，到隔壁暖和的实验室坐坐，打开电脑，接着饭否发泄，豆瓣上面变态小组转转，骂骂酵母，看看冷笑话，和同学侃侃。

十一点吃午饭。

十二点回实验室，空调房间边吹边睡觉。睡醒了继续饭否，豆瓣。外加东转西转，有事聊天，没事下载。

十四点终于可以面对导师和学长，虚情的问问要不要帮忙，假意的在心里念你肯定不放心我所以还是算了吧。写开题，查试剂，总算做会儿人事。

十六点开始酝酿下班，去抽抽烟，聊聊天。

十七点下班，立马狗模人样，公交车，饭堂，回宿舍消遣消遣消遣消遣到第二天。