After digestion has progressed for one to two days another 10 c.c. of liquor pancreaticus may be added.

The products of digestion in one case should be examined, say, after six hours' digestion, and in another case after thirty-six hours' digestion or more. The digestive products should then be searched for; the early products of digestion (alkali-albumin, deutero-proteose, &c.) will become less abundant with the length of time that digestion has been allowed to progress, and the later products (peptone, leucine, tyrosine, tryptophane, &c.) will become more abundant. The methods for testing most of these substances have been already given. The following are the tests for tryptophane, leucine, and tyrosine :

(a) Tryptophane.—Add a few drops of bromine water; a violet colour is produced.

(b) Leucine and Tyrosine.-i. Examine microscopical specimens of these. The deposit generally found in rather old specimens of Benger's liquor pancreaticus will be a convenient source of these crystals.

ii. To some of the pancreatic digest add Millon's reagent and filter off the precipitated protein. Boil the filtrate, and the presence of tyrosine is indicated by a red colour. If tyrosine is abundant the red colour appears without boiling. Leucine does not give this test.

iii. Faintly acidify another portion of the filtered digest with acetic acid and boil; if any protein matter is still undigested it will be thus coagulated and can be filtered off. Reduce the filtrate to a small bulk until it begins to become syrupy. Leave overnight in a cool place, and crystals mainly of tyrosine will separate out. Filter these off through fine muslin, and evaporate down the filtrate to the consistency of a thick syrup; leave this overnight again, and a second crop of crystals, forming a skin on the surface and consisting mainly of leucine, will have separated out.

7. Zymogen Granules.-Examine microscopically, mounting in aqueous humour or serum (or in glycerin after treatment with osmic acid vapour), small pieces of the pancreas, parotid, and submaxillary glands in a normal guinea pig,' and also in one in which profuse secretion had been produced by the administration of pilocarpine.

Note that zymogen granules are abundant in the former, and scarce in the latter, being situated chiefly at the free border of the cells.

Extremely good, though not permanent, microscopic specimens may be obtained by teasing in a 33-per-cent. solution of caustic potash.

'The guinea-pigs should be killed by bleeding, and the blood collected and defibrinated, and utilised for the preparation of oxyhæmoglobin crystals. This will give students an opportunity of seeing the exceptional form (tetrahedra) in which the blood-pigment of this animal crystallises.

The three methods of obtaining crystals described on p. 112 all give good results. If amyl nitrite is used instead of ether in the third method, crystals of methæmoglobin are obtained.

LESSON XIX

HEMOGLOBIN AND ITS DERIVATIVES

Defibrinated ox-blood suitably diluted may be used in the following experiments as in those described in Lesson IX.

1. Place some in a hæmatoscope (see fig. 33, p. 116) in front of the large spectroscope. Note the position of the two characteristic bands of oxyhæmoglobin; these are replaced by the single band of hæmoglobin after reduction by the addition of Stokes's reagent (see footnote, p. 115) or ammonium sulphide. By means of a small rectangular prism a comparison spectrum showing the bright sodium line (in the position of the dark line named D in the solar spectrum) may be obtained, and focussed with the absorption spectrum.

2. Obtain similar comparison spectra by the use of the microspectroscope. For this purpose a cell containing a small quantity of oxyhemoglobin solution may be placed on the microscope stage, and a test-tube containing carbonic oxide hæmoglobin in front of the slit in the side of the instrument. Notice that the two bands of carbonic oxide hæmoglobin are very like those of oxyhæmoglobin, but are a little nearer to the violet end of the spectrum.

Carbonic oxide hæmoglobin may be readily prepared by passing a stream of coal gas through the diluted blood. It has a cherry-red colour and is not reduced by the addition of ammonium sulphide (fig. 57, spectrum 4).

3. Methæmoglobin.--Add a few drops of ferricyanide of potassium to dilute blood and warm gently. The colour changes to mahogany-brown. Place the test-tube in front of the small direct-vision spectroscope. Note the characteristic band in the red (fig. 57, spectrum 5). On dilution other bands appear (fig. 57, spectrum 6). Treat with ammonium sulphide and the band of hæmoglobin appears.

4. Acid Hæmatin.—(a) Prepare the following mixture:-150 c.c. of 90-percent. alcohol and 6 c.c. of concentrated sulphuric acid; take about 5 c.c. of this mixture and boil it in a test-tube. While still hot drop into it a few drops of undiluted defibrinated blood, and filter. Note the brown colour of the filtrate. Compare the position of the absorption band in the red with that of methæmoglobin; that of acid hæmatin is further from the D line (fig. 57, spectrum 7).

(b) Add some glacial acetic acid to undiluted defibrinated blood. Extract this with ether by gently agitating it with that fluid. The ethereal extract should then be poured off and examined spectroscopically. The band in the red is seen, and on further diluting with ether three additional bands appear. 5. Alkaline Hæmatin.—(a) Add to diluted blood a small quantity of strong

FIG. 57.-1, Solar spectrum. 2, Spectrum of oxyhæmoglobin (0.37 p.c. solution). First band, à 589564 second band, A 555-517. 3, Spectrum of hæmoglobin. Band, A 597-535. 4, Spect um of COhæmoglobin. First band, A 583-561; second band. A 547-521. 5, Spectrum of methæmoglobin (concentrated solution). 6, Spectrum of methæmoglobin (dilute solution). First band, X 647622; second band, A 587-571; third band, A 552-532; fourth band, à 514-490. 7, Spectrum of acid hæmatin (ethereal solution). First band, à 656-615; second band. A 597-577; third band, a 557-529 fourth band, A 517-488. 8, Spectrum of alkaline hæmatin. Band from λ 630-581. 9, Spectrum of hæmochromogen (reduced hæmatin). First band, A 569-542; second band, A 535-504. 10, Spectrum of acid hæmatoporphyrin. First band, à 607-593; second band, A 585536. 11, Spectrum of alkaline hæmatoporphyrin. First band, A 633-612; second band, A 589-564; third band, A 549-529; fourth band, à 518-488. The above measurements (after MacMunn) are in millionths of a millimetre. The liquid was examined in a layer 1 centimetre thick. The edges of ill-defined bands vary a good deal with the concentration of the solutions.

Methæmoglobin

Oxyhæmoglobin

очн

caustic potash and boil. The colour changes to brown, and with the spectroscope a faint shading on the left side of the D line is seen (fig. 57, spectrum 8).

(b) The band is much better seen in an alcoholic solution. Prepare the following mixture:-150 c.c. of 90-per-cent. alcohol, and 18 c.c. of 50-percent. potash. Take about 5 c.c. of this mixture in a test-tube and boil it. G h HK L M N

Hb

Solar spectrum

FIG. 59.-The photographic spectrum of oxyhæmoglobin and methæmoglobin. (Gamgee.)

While still hot drop into it a few drops of undiluted defibrinated blood. The fluid then shows the spectrum of alkaline hæmatin. This may then be used for the next experiment.

6. Hæmochromogen.-Add ammonium sulphide to the solution of alkaline hæmatin; the colour changes to red, and two bands are seen, one between D

and E, and the other nearly coinciding with E and b (fig. 57, spectrum 9). The spectrum of alkaline hæmatin reappears for a short time after vigorous shaking with air.

7. Hæmatoporphyrin.-To some strong sulphuric acid in a test-tube add a few drops of undiluted blood, and observe the spectrum of acid hæmatoporphyrin (iron-free hæmatin) (fig. 57, spectrum 10). Map out all the spectra you see on a chart.

8. The photographic Spectrum. Hæmoglobin and its compounds also show absorption bands in the ultra-violet portion of the spectrum. This portion of the spectrum is not visible to the eye, but can be rendered visible by allowing the spectrum to fall on a fluorescent screen, or on a sensitive photographic plate. In order to show absorption bands in this part of the spectrum very dilute solutions of the pigment must be used.

In order to demonstrate these bands, the telescope of a large spectroscope is removed, and a beam of sunlight or of light from the positive pole of an arc lamp is allowed to fall on the slit of the collimator. The spectrum is focussed on a fluorescent screen.1 The slit is then opened very widely, and the coloured solution is interposed on the path of the beam falling on the slit.

Oxyhemoglobin shows a band (Soret's band) between the lines G and H. In hæmoglobin, carbonic oxide hæmoglobin, and nitric oxide hæmoglobin, this band is rather nearer G. Methæmoglobin and hæmatoporphyrin show similar bands.

The two preceding figures show the 'photographic spectra' of hæmoglobin, oxyhemoglobin, and methæmoglobin, and will serve as examples of the results obtained. I am greatly indebted to Prof. Gamgee, to whom we owe most of our knowledge on this subject, for permission to reproduce these two specimens of his numerous photographs.

9. Preparation of Pure Oxyhemoglobin.—The following method is described in Stirling's 'Practical Physiology.' Centrifugalise dog's defibrinated blood and pour off the serum. Centrifugalise again with physiological saline solution repeatedly until the supernatant fluid contains only traces of protein. Mix the magma of corpuscles with two or three volumes of water saturated with acid-free ether; the solution becomes clear. Then add a few drops of 1-per-cent. solution of acid sodium sulphate till the mixture looks tinted like fresh blood, owing to the precipitation of the stromata. These can be separated by filtration. Pour off the clear red fluid: cool it to 0° C., add one-fourth of its volume of absolute alcohol previously cooled to 0° C. Shake well, and then let the mixture stand at 5°-15° C. for 24 hours. As a rule the whole passes into a glittering crystalline mass. Filter at 0° C. and wash with ice-cold 25-per-cent. alcohol. Redissolve the crystals in a small quantity of water, and recrystallise as before. The crystals may then be spread on plates of porous porcelain, and dried in a vacuum over sulphuric acid.

Fluorescent screens, similar to those in common use in observations made with Röntgen rays, may be made by coating white cardboard with barium platinocyanide.