Fluorescence lifetimes in hydrated bovine serum albumin powders

Vol. 114, No. 3, 1983 August

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 901-906

12, 1983

FLUORESCENCE LIFETIMES IN HYDRATED BOVINE SERUMALBUMINPOWDERS George Department Received

June

F. Sheats*

of Chemistry,

and Leslie

University

S. Forster

of Arizona,

Tucson,

AZ 85721

6, 1983

The average relaxation time for tryptophan excited state decay increases progressively with water content in bovine serum albumin powders. A sharp lifetime increase is observed at low water coverage, followed by a slower increase at intermediate hydration levels. As the water the lifetime increase is content exceeds 0.5g H20/g protein, again steep. The been

effect

extensively

physical is

of

hope help

scale

that to

biological

case

studied

and enzyme

the

will

which of

water

of

information

Burstein

have

the

concluded

teins.

Measurement

effect

of adsorbed

of water

the

hydration

on direct

in

effect

proteins,

including

and

of

width

increased

the

lifetimes

is

water-tryptophan

to

in this solid

area

proteins

lo-l1

under time s in

proteins

are

in

emission

the the with

the

nanosecond

domain.

of

hydration

on

bovine

the

in both

proteins

tryptophan in

has

a characteristic

in

dynamics the

fluorescence

and

exchange,

change

examined

position

that

the

work in

water

lifetimes

about

in several

for

with

hydrogen

and determination

determined

and

e.g.,

changes

of hydration

associated

Tryptophan

of tryptophan

They

spectrum

days,

and films

monitoring

between

relaxation.

provides

and

fluorescence

from

effect

is

powders

motivation

interactions

Each property

can vary

content

(3).

the

the

protein

include

A major

about

understand

of

studies

(2).

information

range

properties

These

properties

dielectric

Permyakov

on the (1).

conditions.

nanosecond

tein

water

steady

serum albumin state

flexibility

of

another

emission the

way to probe

interactions

the

prothe

and on pro-

dynamics.

*Permanent

address:

Department New York

of Chemistry, at Plattsburgh,

State University of Plattsburgh, N.Y. 19201 0006-291X/83

901

$1.50

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 114, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

EXPERIMENTAL

RESEARCH COMMUNICATIONS

METHODS

Sample Preparation - Bovine serum albumin (Sigma Chemical Co.) was dried over P2O5 under vacuum for 75 hours before use. A sample was prepared by packing powder into a lmm recess in a brass plate which was then inserted into a quartz fluorescence cuvette. The cuvette and a larger weighed portion of powder were then placed in a closed container together with a beaker of an H2SO4 solution of appropriate composition to control the water vapor pressure. After equilibration for at least 60 hours, the cuvette was sealed with parafilm and a soft rubber cap. The water vapor pressure in the cuvette was maintained constant by placing some of the H2SO4 solution in a 3mm tube within the cuvette. The amount of adsorbed water was determined by weighing the preweighed control and assuming that the sample and control hydration were the same. This procedure was necessary since the sample size was too small for precise estimation of the small increase in weight that was due to the adsorbed water. After the fluorescence measurements were completed, the cuvette was opened and placed in contact with water at a different vapor pressure. In this way the same sample could be used to collect data at several different hydration levels. Data Collection and Analysis - Emission from a powder sample and a reference solution of p-terphenyl in degassed cyclohexane was excited by a thyratronThe radiation was gated flash lamp operated at about l/2 atm N2 pressure. passed through an 0.25 m Jarrell-Ash monochromator and an interference filter to produce 296 nm light. The emission of both the sample and the reference was passed through a 350 nm interference filter. Scattered light was much less than 1% of the sample emission. Conventional time - correlated monophoton counting instrumentation was used to record the time course of the emisson. To minimize the effect of fluctuations in the light source shape during the time required for data collection, 30-200 minutes, the sample and reference were rotated alternately into the excitation beam every 20 s. The emission profile is a convolution of the excitation function E(t) and the decay function, F(t) = laiexp(-t/rf). Only when the data quality is extremely good is it possible to extract ai and ri for even a three-term decay function. We used an iterative convolution least squares method with a twoterm decay function. E(t) was obtained by determining the instrument response function that would yield the correct p-terphenyl lifetime (4). The number of tryptophan environments in a hydrated sample will probably be very large and the analytical results are very sensitive to noise and/or lamp fluctuations. This leads to variation in the ~1, r2, and al/a2 values. It has been found that the average lifetime for the emission of organic molecules adsorbed on silica gel, ? = (alr12 + a2r22)/(alrl + a2T2), remains constant for a particular sample, even when the individual parameters vary from run to run for a given sample (5). In this work = alrl + a272 (al + a2 = 1) and, indeed, the individual parameters were quite reproducible for repeated runs with the same sample, but ? exhibited much less scatter for different samples equilibrated against the same water vapor partial pressure. Therefore, ? was the quantity used to monitor the effect of hydration. RESULTS AND DISCUSSION The used

results

and

these

atmospheres. without

a given

powder B.

in

were

cycled

In a number

twofold

Sample

are.listed

In

the

sample

the

A total

of seven

through

of water

r.

content 902

time

h = g H2O/g

increasing

the

powder

different

the equilibration

affecting

direction B,

I.

sequentially

of cases

appreciably in

table

samples water

was varied

protein

water

was progressively

vapor

more than

was changed

content,

was

except

decreased

for for from

Vol. 114, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I- 7 (ns) AT DIFFERENT HYDRATION LEVELS IN BOVINE SERDM ALRUMIN h

0.02

0.05

0.08

0.11

0.16

0.18

0.29

0.41

0.59

A

3.05

3.41

3.45

3.67

3.70

3.74

3.93

4.02

4.33

B

3.10

3.61

3.64

3.63

Sample

C

3.33

D

3.33

3.60

3.77 3.51

E

3.77

4.03

3.54

4.65

3.86

3.93

F

4.08

4.61

G

h=0.18

to

water

vapor

above, are

4.67

h=0.02. pressure

the 7 values quite

water

for

yielded

in Table The

fig.1.

Three

I.

certain.

the

following

h=O.lS-0.18,

uncertainty regions

limit in

the

I

h in

does

value

of

obscure

I 0.4

by a given dif-

with

values

of h are

was 0.02. basic

small

I

solution

h=0.40-0.42,

Average

the (i)

I

Q'S04

h produced

powder

trend

exhibited

hydration,

h

<

I 0.E i

h (g HzO/g protein) Figure

1 -

The average lifetime, ferent hydration

r=

levels

(al~l*

in bovine 903

each

As described

h=0.56-0.60,

the driest

I 0.2

fig.1.

at

determinations,

results:

not

values

a particular

Duplicate

may be distinguished:

0

Mean

h=0.067-0.09.

for

h

over

of

less

The upper

of

equilibrated

assignment

is

of hysteresis.

as a function

samples

h=O.lS-0.21,

h=0.28-0.30,

no evidence

shown

The

pressure

powders

is

are

reliable.

vapor

ferent

used

There

+ a2T2*)/(alTl

serum albumin.

+ a2’2),

at

dif-

in 0.10,

BIOCHEMICAL

Vol. 114, No. 3, 1983

where

7

increases

rapidly

increases

slightly;

increases

markedly, The

The

spectral value

powder

emission

spectrum.

is

data

of the

other

The

good

(Table

I)

from

interior

surface

phenomena Tryptophan

that

is

solution,

quenchers

points

being

on the

molecules water exciplex

cannot

effectively. bovine

to form.

is In are

v&.,

By surface and not this

from

the

species, the molecu-

effect

might

results

could

be very

different

difpowder

that

the

high

to sample

emission

follows,

we will

sufficiently discussion

not

illumination,

respect,

hydration

on the

of h.

pulsed

studies,

indicates

the

solution

arises

an exciplex. with

that

important

surface lifetime

and

incident

that

the

where

they

the

light

only

there

molecule.

during the

are Thus,

of the

protein

of

the

(7,A). excited

possible

for

water

lifetime,

molecules

it

proximate

state

Hz0

400

albumin

powders,

must

to

serum

In

water

maxi-

tryptophans

with

about

904

the

can interact state

species

fluorescence

in bovine

adsorbed

is

solvated

residues

excited

tryptophan

In addition,

a strongly

accessibility

tryptophan

of

from

The position

the

to both

When h=O.l

serum albumin

vary,

layers

in

molecular

migrate

or

the

emission

of water

values

species.

the

The but

solution

important

and bulk

(h=0.62).

involved.

strongly

the

one

between

effects

coupled

during can

are

steady

surface

surface

surfaces

surface

likelihood

mum in

the

the

emission

in all

at intermediate

In hydration

molecules.

powder

effect

molecules.

reproducibility where

the

of the crystallites

since

protein

that

from

the

the surface

the subsurface

assume

spectrum

of h.

spectrum,

than

again

values

344nm

solution

about

in

ferent.

into

quantitatively

methods

protein

the

T

7

ns (6).

different

to

broader

spectroscopic

serious

penetration

in

either

near

at

where

where

7 = 6.51

nm (hnO.05)

distinctly

emission

hO0.5)

recorded

under

be especially

samples

all

emission

toward

of the

336

intermediate,

value,

measurements,

may be biased

surface

also

(ii)

content

345 nm value

can be said

we mean the molecules lar

from

are

water

were

RESEARCH COMMUNICATIONS

water;

to the solution

shifts

spectra

Emission from

high

spectra

to the

or shape

differ

(iii)

close

Little

position

adsorbed

presumably

maximum

latter

with

and

emission

AND BIOPHYSICAL

rotate

exciplexes

if

no the

to interact adsorbed

on

to form.

a

BIOCHEMICAL

Vol. 114. No. 3, 1983 There

are

dielectric NMR

constant

linewidth

but

are

(10)

change

identical

at

all

correlation

hydration.

The

relaxation

h=O

is

lysozyme

(2),

tempting

to ascribe

to

that

emitters

water

increasing

The

variation

emission

complement

determinations

times

been

extensively of protein

prove which

tryptophan

powders

for

the

h=0.2

is

which

*,

protein

in

which

general bound

to

increase changes

the

interactions. increase

water

the

rates

emission

increase

below

interact

with

proteins

with

will

lifetimes

of

properties,

examined

with

lifetime

in this

a measure

of global

a model

tryptophan

with

the

longitudinal

lifetime

does not

that

instructive is

and

accord

tryptophan

in

increasing

with

water-tryptophan

probability of

lysozyme

below

to compete the

and

structure

addition

sufficiently

the

should

direct

with

albumin

fluorescence

of the protein

than

indicate

the

at

protons

with

adsorbed

limit

water

transverse

In

a

The

for

molecules

(1,12).

albumin.

to a decrease

interpreted

water

interpretation,

reflects

in studies

were

the

points

proton

and

monotonically

widths

water

(9)

reaches

serum

line

hydration

with

motions

bovine

adsorbed water

serum

and

The NMR line

resonance

the

rather

tryptophan.

have

in

of bovine

GHz increases

both

the

environment,

simply

buried

of

NMR linewidth

h=O.l

at 9.95

of h for proton

RESEARCH COMMUNICATIONS

lifetfme,

to

at h=O.l.

to a loosening

protein

up

constant

and -0.1

Tryptophan

useful

steep

the

In an alternate

excited only

is

hydration

and it

internal

hz0.5

content

hydrated

the NMR data

However,

(12).

water

increases

protein

groups

tryptophan

of

the

time

for

motion of

between

with

changes

times

water

polar

of

values of

rotational

picture

change

accelerates

The narrowing

the

the

The dielectric

the

(9).

in

decrease

h=0.15-0.20. h,

parallels

AND BIOPHYSICAL

in

context.

a local

property,

NMR.

serves

Fluorescence

solutions.

to

life-

They will

prove

as well.

ACKNOWLEDGMENTS This

research

(GM30017).

was supported We are

grateful

by a grant

from

to Dr.

Rupley

J.A.

the National for

very

Institutes helpful

of Health discussions.

REFERENCES 1. Kuntz, 2. Rupley, 18-22.

I.D., J.A.,

and Kauzmann, Gratton, E.,

W. (1974) and Careri,

905

Adv. Protein Chem. 28, G. (1983) Trends Biochem.

239-345. Sci. 8,

Vol. 114, No. 3, 1983

BlOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

3. Permyakov, E.A. and Burstein, E.A. (1977) Stud. Biophys. 64, 83-93. 4. Wahl, Ph., Auchet, J.C., and Donzel, B. (1974) Rev. Sci. Instrum. 45, 29-32. 5. Borenstein, R., deMayo, P., Okada, K., Rafalsha, M., Ware, W.R., and Wu., K.C. (1982) J. Amer. Chem. Sot. 104, 4635-4644. 6. Grinvald, A. and Steinberg, I.Z. (1976) Biochim. Biophys. Acta 427, 663-678. 7. Burstein, E.A., Vedekina, N.S., and Ivkova, M.N. (1973) Photochem. Photobiol. 18, 263-279. 8. Eftink, M.R. and Ghiron, C.A. (1976) Biochemistry 15, 672-680. 9. Fuller, M.E. and Brey, W.S. (1968) J. Piol. Chem. 243, 274-280. 10. Gascoyne, P.R.C., and Pethig, R. (1981) J. Chem. Sot. Faraday Trans 1 77, 1733-1735. 11. Hilton, B.D., Hsi, E., and Bryant, R.G. (1977) J. Am. Chem. Sot. 99, 8483-8490. 12. Shirley, W.M., and Bryant, R.G. (1982) J. Am. Chem. Sot. 104, 2910-2918.

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