STUDIES ON HEMOGLOBIN. III An Ultra-Micro-method for the Determination of Hemoglobin as a Peroxidase.

Transcription

1 BY STUDIES ON HEMOGLOBIN. III An Ultra-Micro-method for the Determination of Hemoglobin as a Peroxidase. HSIEN WU (From the L' b)oratory of Physiological CCemistry. Peking Union Medical College, Peking) (Received for Publication, October 10, 1922) Several excellent methods far the determination of the hemo globin in blood are already available. A new method must consequently possess unusual merits to justify its introduction. The method to be described may truly be called an ultra-micro method, for as little hemoglobin as is contained in one ten thousandth of a cubic centimeter of blood suffices for a determina tion. It is therefore well suited for the st.idy of the hemoglobin in the blood of small animals and embryoes to which none of the existing methods is applicable. The usefulness of the new method is not limited to the blood. With slight modifications it should prove applicable to the determination of hemoglobin in tissues and body fluids other than the blood. PRI\CI1 LE. The method is based on the comparison of the brown color produced from benzidine in acetic acid solution by the simultane ous action of the hemoglobin and hydrogen peroxide. It is in fact a method for the determination of a " pseudo-peroxidase," but whether it can be applied without modification to the deter mination of true plant and animal peroxidases must be reserved for future study. The new method is as follows :

2 190 Hsien Wu : SOLUTIONS REQUIRED. 1. Benzidine. Dissolve 2 grams of high grade benzidine in 20 cc. of glacial acetic acid with the aid of warming. Cool. Add 80 cc. of water and 0.5 to 1 gram of blood charcoal. Shake gently for 10 to 15 minutes and filter. The charcoal should remove nearly all the brown substance always present in com mercial benzidine, and the filtrate should be almost colorless or at most faintly tinted. In brown bottles the solution remains serviceable for 1 to 2 weeks, but the darkened can be reclaimed by treating again with charcoal. 2. Hydrogen peroxide. The commercial 3 per cent solution is used. It should be titrated with standard potassium permanganate solution to make sure that no extensive deterioration has occured. 3. Standard hemoglobin solution. Any of the standard hemo globin solutions recommended in the first paper of this series can be used after proper dilution. 4. Acetic acid. A 20 per cent solution made by mixing 1 volume of glacial acetic acid with 4 volumes of water. PROCEDURE. Dilute 1 cc. of blood to 100 cc. Measure 1 cc. of the resulting solution and dilute again to 50 cc., giving a 1:5000 solution. Dilute also 1 cc. of the stock standard solution to 500 cc. if it is a 10 per cent blood solution or to 50 cc. if it is a one per cent solution. Measure 2 cc. of the benzidine solution into each of two clean and dry test tubes graduated at 25 cc. Then add 1 cc. of the standard hemoglobin solution to one tube and 1 cc. of the diluted blood solution to the other. Add 1 cc. of hydrogen peroxide to each and mix. After 15 minutes make up to volume with 20 per cent acetic acid, stopper with a clean rubber stopper, mix and read in the colorimeter. The calculation is very simple. The reading of the standard (at 20 mm.) divided by the reading of the unknown, and the result multiplied by the percentage of the hemoglobin present in the

3 Studies on Hemoglobin. 191 blood from which the stock standard was prepared gives the per cent of the hemoglobin in the unknown blood. The close agreement between the results obtained by the new method and those obtained by the colorimetric methods is shown in Table I of the preceeding paper. REMARKS ON THE BENZIDINE REACTION. Under the conditions described above the maximum color of the peroxidase reaction is reached in about 25 minutes, the hemoglobin being all destroyed by the peroxide at the end of this time. Although the color continues to grow very gradually due to the oxidation of the benzidine by the peroxide alone, the effect of this oxidation on the relative color values is negligible if the solutions are not allowed to stand for more than half an hour. The readings taken at the end of 30, 20, 10 or even 5 minutes are almost exactly proportional to the concentrations of the hemoglobin within limits corresponding to 1:2500 and 1:10000 dilutions of normal blood containing 14 per cent of hemoglobin when the 1:5000 dilution is used as the standard (Table I). Within narrower limits usually adopted in colorimetry, that is, when the unknown and the standard are not more than 30 per cent apart the readings are exactly proportional to the concentrations. We recommend a standing of 15 minutes, because this seems to be a convenient duration of time to insure uniform TABLE I. Readings of color produced by the action of hydrogen peroxide and varying dilutions of blood on benzidine.

4 192 Hsicn Wu : conditions of manipulation for a set of three or four determinations. The amounts of color produced by equivalent amounts of oxyhemoglobin, methemoglobin, carboxyhemoglobin, and cyan hemoglobin are the same. But a corresponding amount of hematin gives only about 70 per cent of the color given by the chromoproteins.* If to 1 cc. of 1:5000 blood solution a drop of glacial acetic acid is added followed after a few minutes by the benzidine solution and hydrogen peroxide the color developed is only 70 per cent of that obtained when the acetic acid is not added or added after the benzidine solution has been added. The acetic acid has split off the hematin from the hemoglobin. But the blood solution may be mixed with the benzidine solution, which is 20 per cent in acetic acid, and allowed to stand for 10 minutes or longer without any loss of activity. The benzidine exerts a protective action on the hemoglobin. This protection is afforded not through the reduction of the acidity by the benzidine, for sodium acetate has no such protective action. In the light of the modern theory of enzyme action we would suggest as an explanation of this phenomenon that the hemoglobin (enzyme) combines with the benzidine (substrate). The following protocoll of an experiment with 1:5000 blood may be cited. (1) 1 cc. blood solution cc. benzidine + 1 drop acetic acid + 1 cc. peroxide as standard set at 20 into. (2) 1 cc. blood solution + 1 drop acetic acid, stood 5 min cc. benzidine + 1 cc. peroxide reads 28. (3) 1 cc. blood solution + 11/2 cc. benzidine, stood 10 mins. + 1 cc. peroxide reads 20. (4) 1 cc. blood solution + 2 drops 10 per cent sodium acetate + 11 cc. benzidine + 1 drop acetic acid + 1 cc. peroxide reads 17. (5) 1 cc. blood solution + 2 drops 10 per cent sodium acetate + 1 drop acetic acid, stood 5 mins. + 1 cc. benzidine + 1 cc. peroxide reads 25. * See the preceeding paper.

5 Studies on Hemoglobin. 193 It will be noted that the amount of the color is increased by the addition of sodium acetate, but the proportional loss of activity is just the same as when no acetate is added before the addition of acetic acid. Owing to the ease with which the hemoglobin is attacked by the acid we have adopted a procedure of adding the blood solu tion to benzidine and not the reverse. There is in this way at all times an excess of benzidine to combine with the hemoglobin and absolutely no loss of activity can occur. If the benzidine is added to the blood there may be at the first moment not enough benzidine to combine with the hemoglobin and yet sufficient acidity to split off some hematin. We have found indeed that when the benzidine solution is added to a row of several tubes each containing 1 cc. of the same dilute blood solution and then 1 cc. of peroxide is added to each tube, the amounts of color developed in different tubes show at times a variation of 5 per cent or even more which cannot bo explained in any way other than that suggested. When blood is added to the benzidine solution no such irregularity occurs. The amount of color developed depends not only on the con centration of the hemoglobin but also on that of the benzidine. Other things being equal, the color is roughly proportional t ) the concentration of the benzidine in the reaction mixture. With 1 cc. of blood solution, 1 cc. hydrogen peroxide and 1.6, 1.8, 2.0, 2.2, 2.4, cc. of the benzidine solution the colorimetric readings are respectively 20, 18.7, 17.7, and 16.1, using the first solution as the standard. Hence the benzidine solution should be measured with as much care as the blood solution. The amount of hydrogen peroxide has but little influence on the color. The color developed at the end of 20 minutes with 1 cc., 0.75 cc., and 0.5 cc. of peroxide are practically the same, with 0.25 cc. the color is 10 per cent too weak. By increasing the amount of the benzidine solution the amount of blood required for a determination may be reduced to a fraction of that recommended in this paper. The color may be

6 194 Hsien Wu. further increased by using a hydrogen peroxide solution contain ing 5 per cent of sodium acetate. But as the amount of blood required is already so small nothing is gained by further reduc tion especially as benzidine is not a cheap chemical. Owing to the high sensitiveness of the reagent and the exceed ingly small amount of blood dealt with in the reaction, it is needless to say that all contaminations must be strictly excluded. We have found it convenient in our work to place aside a set of pipettes, flasks, graduated tube and rubber stoppers each of which is assigned a specific function and is not used for any other purpose. We also make our determinations always in duplicate, for only in this way can a slight contamination be discovered. SUMMARY A method has been worked out for the determination of minute quantities of hemoglobin.