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Characteristics of high-affinity folate binding in serum of a patient with chronic myelogenous leukemia
383
Clinica Chimica Acta, 105 (1980) 383-366 0 Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 1445
CHARACTERISTICS OF HIGH-AFFINITY FOLATE BINDING IN SERUM OF A PATIENT WITH CHRONIC MYELOGENOUS LEUKEMIA
JAN HOLM *, STEEN INGEMANN HANSEN and JBRGEN LYNGBYE Department (Denmark)
of Clinical Chemistry, Research Division, Central Hospital, DK 3400 Hilleroed
(Received February 2nd, 1980)
Introduction We have recently shown that it is possible to distinguish between a high- and a low-affinity type of folate binding in serum from healthy male subjects [ 11. The high-affinity binder which was a trace protein (M, - 35 000) eluted with 30 mmol/l NaCl (pH 6.3) in the early effluent of serum after DEAE-Sepharose CL-6B chromatography [ 11, while low-affinity binding activity mainly associated with albumin eluted with 1 mol/l NaCl [ 11. The present study describes important qualitative and quantitative deviations from the normal pattern of high-affinity serum folate binding in a patient with chronic myelogenous leukemia. Materials and methods [3H]folate with a specific activity of 65 Ci/mmol was supplied by the Radiochemical Centre, Amersham, U.K. All methods and experimental procedures employed in the present study have recently been described in detail in this journal [ 11. Venous blood samples were drawn from a 55-year-old man with chronic myelogenous leukemia (white cell count > 250 X 109/1 and a peripheral blood smear completely predominated by promyelocytes, myeloblasts as well as stem cells) shortly after his admission to the hospital, and prior to any medical treatment.
* To whom correspondence should be addressed.
384
L”
.
05 Bound
IO folote nmol/l
--+Y-45
47
Fig. 1. Scatchard plot of folate binding to serum. Equilibrium dialysis experiments at 37’C (single determinations) in 0.17 mol/l Tris buffer, pH 7.4. High-affinity binding, (maximum folate binding given by the intersection on abscissa). Low-affinity binding, - - - - - -.
Results and discussion The folate binding pattern in the serum of a patient with chronic myelogenous leukemia was studied in equilibrium dialysis experiments (pH 7.4, 37”C), and the data were analysed in a Scatchard plot (Fig. 1). The biphasic appearance of the plot indicates the presence of a high- (maximum folate binding 1.0 nmol/l) and a low-affinity type of binding. Furthermore, the downward concavity of the first part of the curve consisting of high-affinity binding data is compatible with the involvement of at least two co-operatively interacting sites in binding (Fig. 1). A Hill plot of the same data (Fig. 2) confirmed that highaffinity folate binding displays positive co-operativity, the Hill coefficient being
Fig. 2. Hill plot of high-affinity folate binding cient (the slope of the line) indicated by n.
data from
Fig. 1 (fractional
folate binding.
8). Hill coeffi-
385
IO
Elution
volume
(ml)
Fig. 3. Folate binding profile in effluent obtained from serum after DEAE-Sepbarose CL-6B anion exchange chromatography. The column (2.0 cm2 X 37 cm) was eluted with imidazole buffer, PH 6.3, in a NaCl gradient. Arrow indicates a rise in NaCl concentration from 30 mmol/l to 1 mol/l. Equilibrium dialysis experiments with 0.01 nmol/l (O- - - - - -0). 0.10 nmol/l (A. . . * . -A),and 1.0 nmol/l (g-----.) C3Hl folate.
1.42 + 0.04 (this value is significantly higher than 1.00, p < 0.001). The association constant for high-affinity binding, Kass(the reciprocal of the folate concentration at half saturation) was 3 * 10” mol/l (Fig. 2). Protein binding of folate (0.01-1.0 nmol/l) was determined in effluent fractions collected after passage of serum through a DEAE-Sepharose@ CL-6B column [l]. The binding profile (Fig. 3) reveals large amounts of high-affinity binder in early effluent eluted with 30 mmol/l NaCl, and low-affinity binding activity in fractions eluted with 1 mol/l NaCl. As previously shown, the latter fractions contained large amounts of albumin [ 11. The molecular size of the high-affinity binder in the early effluent was 25 000 as determined by gel filtration [l]. To conclude, characteristics of high-affinity folate binding in serum from a patient with chronic myelogenous leukemia were found to deviate from those in serum from a group of healthy control subjects [l] in the following respects. Firstly, the high-affinity binding activity was much higher (Figs. 1 and 3) than in serum from normal subjects [ 11. Secondly, the molecular size of the highaffinity binder in the patient’s serum (M, - 25 000) was smaller than that of the high-affinity binder in serum from normal subjects (M, - 35 000) [ 11. Thirdly, the high-affinity binding in the patient’s serum displayed positive co-operativity (Figs. 1 and 2), whereas this was not the case in serum from control subjects [ 11. The latter interesting difference in mechanism of high-affinity binding is difficult to explain, but it could be related to the raised level and/or the smaller molecular size of the high-affinity binder in the patient’s serum. However, there is also a possibility that the patient’s serum contains a principle or co-factor moderating the interaction between folate and the binding sites on the protein. In fact, we have recently shown that a co-factor with these characteristics is present in cow’s milk [2], and that the positive coaoperativity characterizing the high-affinity folate binding system in cow’s milk [3] disappears
336
in the absence progress.
of the co-factor
[2].
Studies dealing with this problem
are in
Acknowledgements The valuable technical assistance of Mrs. J. Rasmussen and Mrs. S. Nordlunde is acknowledged. The study was supported by grants from the Bryde Nielsen Foundation and the Danish Medical Research Council (512-15528). References 1 Helm, J., Hansen, S.I. and Lyngbye, J. (1980) 2 Hansen. S.I., Holm, J. and Lyngbye, J. (1979) 3 Hansen, S.I., Helm, J. and Lyngbye, J. (1978)
Clin. Chim. Acta 100, 113-119 Biochim. Biophys. Acta 579.479-482 Biochim. Biophys. Acta 535, 309-318
Clinica Chimica Acta, 105 (1980) 383-366 0 Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 1445
CHARACTERISTICS OF HIGH-AFFINITY FOLATE BINDING IN SERUM OF A PATIENT WITH CHRONIC MYELOGENOUS LEUKEMIA
JAN HOLM *, STEEN INGEMANN HANSEN and JBRGEN LYNGBYE Department (Denmark)
of Clinical Chemistry, Research Division, Central Hospital, DK 3400 Hilleroed
(Received February 2nd, 1980)
Introduction We have recently shown that it is possible to distinguish between a high- and a low-affinity type of folate binding in serum from healthy male subjects [ 11. The high-affinity binder which was a trace protein (M, - 35 000) eluted with 30 mmol/l NaCl (pH 6.3) in the early effluent of serum after DEAE-Sepharose CL-6B chromatography [ 11, while low-affinity binding activity mainly associated with albumin eluted with 1 mol/l NaCl [ 11. The present study describes important qualitative and quantitative deviations from the normal pattern of high-affinity serum folate binding in a patient with chronic myelogenous leukemia. Materials and methods [3H]folate with a specific activity of 65 Ci/mmol was supplied by the Radiochemical Centre, Amersham, U.K. All methods and experimental procedures employed in the present study have recently been described in detail in this journal [ 11. Venous blood samples were drawn from a 55-year-old man with chronic myelogenous leukemia (white cell count > 250 X 109/1 and a peripheral blood smear completely predominated by promyelocytes, myeloblasts as well as stem cells) shortly after his admission to the hospital, and prior to any medical treatment.
* To whom correspondence should be addressed.
384
L”
.
05 Bound
IO folote nmol/l
--+Y-45
47
Fig. 1. Scatchard plot of folate binding to serum. Equilibrium dialysis experiments at 37’C (single determinations) in 0.17 mol/l Tris buffer, pH 7.4. High-affinity binding, (maximum folate binding given by the intersection on abscissa). Low-affinity binding, - - - - - -.
Results and discussion The folate binding pattern in the serum of a patient with chronic myelogenous leukemia was studied in equilibrium dialysis experiments (pH 7.4, 37”C), and the data were analysed in a Scatchard plot (Fig. 1). The biphasic appearance of the plot indicates the presence of a high- (maximum folate binding 1.0 nmol/l) and a low-affinity type of binding. Furthermore, the downward concavity of the first part of the curve consisting of high-affinity binding data is compatible with the involvement of at least two co-operatively interacting sites in binding (Fig. 1). A Hill plot of the same data (Fig. 2) confirmed that highaffinity folate binding displays positive co-operativity, the Hill coefficient being
Fig. 2. Hill plot of high-affinity folate binding cient (the slope of the line) indicated by n.
data from
Fig. 1 (fractional
folate binding.
8). Hill coeffi-
385
IO
Elution
volume
(ml)
Fig. 3. Folate binding profile in effluent obtained from serum after DEAE-Sepbarose CL-6B anion exchange chromatography. The column (2.0 cm2 X 37 cm) was eluted with imidazole buffer, PH 6.3, in a NaCl gradient. Arrow indicates a rise in NaCl concentration from 30 mmol/l to 1 mol/l. Equilibrium dialysis experiments with 0.01 nmol/l (O- - - - - -0). 0.10 nmol/l (A. . . * . -A),and 1.0 nmol/l (g-----.) C3Hl folate.
1.42 + 0.04 (this value is significantly higher than 1.00, p < 0.001). The association constant for high-affinity binding, Kass(the reciprocal of the folate concentration at half saturation) was 3 * 10” mol/l (Fig. 2). Protein binding of folate (0.01-1.0 nmol/l) was determined in effluent fractions collected after passage of serum through a DEAE-Sepharose@ CL-6B column [l]. The binding profile (Fig. 3) reveals large amounts of high-affinity binder in early effluent eluted with 30 mmol/l NaCl, and low-affinity binding activity in fractions eluted with 1 mol/l NaCl. As previously shown, the latter fractions contained large amounts of albumin [ 11. The molecular size of the high-affinity binder in the early effluent was 25 000 as determined by gel filtration [l]. To conclude, characteristics of high-affinity folate binding in serum from a patient with chronic myelogenous leukemia were found to deviate from those in serum from a group of healthy control subjects [l] in the following respects. Firstly, the high-affinity binding activity was much higher (Figs. 1 and 3) than in serum from normal subjects [ 11. Secondly, the molecular size of the highaffinity binder in the patient’s serum (M, - 25 000) was smaller than that of the high-affinity binder in serum from normal subjects (M, - 35 000) [ 11. Thirdly, the high-affinity binding in the patient’s serum displayed positive co-operativity (Figs. 1 and 2), whereas this was not the case in serum from control subjects [ 11. The latter interesting difference in mechanism of high-affinity binding is difficult to explain, but it could be related to the raised level and/or the smaller molecular size of the high-affinity binder in the patient’s serum. However, there is also a possibility that the patient’s serum contains a principle or co-factor moderating the interaction between folate and the binding sites on the protein. In fact, we have recently shown that a co-factor with these characteristics is present in cow’s milk [2], and that the positive coaoperativity characterizing the high-affinity folate binding system in cow’s milk [3] disappears
336
in the absence progress.
of the co-factor
[2].
Studies dealing with this problem
are in
Acknowledgements The valuable technical assistance of Mrs. J. Rasmussen and Mrs. S. Nordlunde is acknowledged. The study was supported by grants from the Bryde Nielsen Foundation and the Danish Medical Research Council (512-15528). References 1 Helm, J., Hansen, S.I. and Lyngbye, J. (1980) 2 Hansen. S.I., Holm, J. and Lyngbye, J. (1979) 3 Hansen, S.I., Helm, J. and Lyngbye, J. (1978)
Clin. Chim. Acta 100, 113-119 Biochim. Biophys. Acta 579.479-482 Biochim. Biophys. Acta 535, 309-318